CN115901659A - Kit and method for detecting stone forming coefficient of urinary calculus - Google Patents
Kit and method for detecting stone forming coefficient of urinary calculus Download PDFInfo
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Abstract
The invention relates to the technical field of urine calculus stone forming coefficient detection, in particular to a urinary calculus stone forming coefficient detection kit and a detection method, wherein the kit comprises an STD reagent and a Test reagent, the STD reagent comprises an oxalic acid solution with the concentration of 40mM, an oxalic acid solution with the concentration of 160mM and a calcium chloride solution with the concentration of 100mM, and the Test reagent comprises a buffer solution with the pH value of 7.4 and a sterile needle filter with the concentration of 0.22 mu m; the core of the kit is that calcium oxalate in urine is activated to form at a limited time and a limited temperature by adding a certain amount of oxalic acid and a sufficient amount of calcium chloride. Let the crystallization fully dissolve after collecting the crystallization, detect solution absorbance with the spectrophotometer, calculate the lithogenesis coefficient of urine through the standard curve, can react the whole equilibrium state of lithogenesis ion and suppression stone ion in the urine to lithogenesis coefficient is as the result, can audio-visually reflect the lithogenesis state and the risk of urine sample.
Description
Technical Field
The invention relates to the technical field of detection of urinary calculus stone forming coefficients, in particular to a kit and a method for detecting urinary calculus stone forming coefficients.
Background
Urinary or kidney stones are a recognized public health problem worldwide. The overall prevalence of the disease varies around the world: 1-5% Asia, 5-9% Europe, 13% North America, and 20% Saudi Arabia. According to literature research, if urinary calculus patients are not effectively prevented and treated after primary treatment, the 3-year recurrence rate of urinary calculus can reach 50%. Urinary stone types are classified according to the main mineral components. Calcium oxalate stones are most common, accounting for 75% of all stone types, other stone types including magnesium ammonium phosphate, ammonium urate, calcium phosphate, uric acid and cystine.
The mechanism of kidney stone formation is quite complex and is not yet fully understood at present. It is well accepted in academia that the initial driving force for kidney stone formation is supersaturation of urine, and saturation is the state in which the lithogenic ions in solution are in equilibrium with their respective salts. Increased excretion of urolithiasis generating ions (also known as stone promoters), such as calcium, oxalic acid and phosphate, and reduction of urine volume and stone inhibitors, such as citrate, potassium and magnesium, will increase free ion activity, which can lead to supersaturation of urine, leading to crystal formation and growth. Many abnormalities of urine, including low urine volume, hyperoxaluria, hypercalciuria, hypouricuria, hyperuricemia, and hypomagnesias, are risk factors for CaOx stone formation.
Currently, there are several approaches that focus on assessing the lithogenic potential in urine, all of which focus more on the saturation of urine. The Born Risk Index (BRI) focuses on the in vitro calcium oxalate crystallization behavior of natural synthetic urine specimens to establish the CaOx crystallization risk index of unprepared natural urine. The method entails slowly heating the urine sample to 37 deg.C, assessing the initial concentration of free calcium by ion selective electrode (RAPIDLab 855 blood gas analyzer), and gradually adding ammonium oxalate solution (40 mmoL/L, 0.5mL per step, 0.75mL per minute) to initiate the formation of CaOx particles in 200mL aliquots of the urine sample. The onset of calcium oxalate crystallisation and the amount of oxalic acid (Ox 2) required to induce the process were measured using a 1101M luminometer with a 585nm filter. The measured values are recorded as BRI values. The method is one of the mainstream methods for measuring the urine saturation at present, but the sensitivity is low, a special high-value instrument is required to observe the time and the state of the initial crystal formation in the urine, the operation steps are complicated, and the observation and the quantification are difficult. In 1998, sriboonlue et al found that oxalic acid can co-precipitate with excess calcium ions and ethanol as a calcium oxalate precipitate at pH 5. This is known as an indirect method of measuring oxalic acid in urine. However, the method cannot directly detect oxalic acid and reflect the saturation state of urine, so that the method is less applied at present. In 1997, grases et al used a principle that unprotected stone surfaces remained in contact with urine, and that contained supersaturated material crystallized on it sooner or later. Therefore, they used this feature to assess the risk of urinary CaOx formation. However, the observation of the crystallization in this experiment is not easy to control, the time required for the experiment is long, and the experiment is not easy to be used in clinical operation.
Disclosure of Invention
The invention aims to provide a detection kit and a detection method for urinary calculus formation Dan Jishu (UCI), which have the advantages of wide detection range, high sensitivity and specificity, simplicity in operation and quickness, and can be used for determining calculus formation potential in urine.
In order to achieve the purpose, the invention adopts the technical scheme that: a kit for detecting the stone forming coefficient of urinary calculus comprises:
a Standard (STD) reagent comprising the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 160mM and calcium chloride solution;
a Test article (Test) reagent, said Test article (Test) reagent comprising the following components: buffer pH 7.4 and 0.22 μm sterile needle filter.
Further: the concentration of the calcium chloride is 100mM/L.
And further: the concentration of the buffer solution is 1M/L, and the diameter of the filter is 20mm.
Further: the buffer was phosphate buffered saline (TBS).
The detection principle of the kit is as follows:
urine lithogenesis index is calculated by measuring the crystals produced by urine over a defined time
Oxalic acid + CaCO 3 - - -CaOx (crystalline) + CO 2 +H 2 O
Further: standard (STD) reagents also include: oxalic acid at a concentration of 60mM, oxalic acid at a concentration of 80mM, or/and grass at a concentration of 120 mM.
As a preferred embodiment of the invention, the kit may further comprise oxalic acid at a concentration of 60mM, oxalic acid at a concentration of 80mM and oxalic acid at a concentration of 120 mM. It is obvious to those skilled in the art that oxalic acid solutions having concentrations of 60mM, 80mM and 120mM may be prepared from oxalic acid solutions of 40mM and 160mM during actual use.
The calcium carbonate solution in the standard Substance (STD) reagent is used for preparing a detection sample and is directly used without dilution.
The buffer solution in the Test reagent is phosphate buffer salt solution, has a pH value of 7.4, is used for washing formed crystals, removes impurities and improves the accuracy of a detection result.
A 0.22 μm sterile needle filter is used to filter large molecules and impurities from urine, typically twice per urine sample.
The method for detecting the calculus forming coefficient of the urinary system calculus by using the kit provided by the invention comprises the following specific steps:
(1) Preparing a standard substance and a detection sample;
the standard substance is as follows: adding 950 mu L of deionized water and 50 mu L of standard oxalic acid solution with different concentrations into a glass test tube, and fully and uniformly mixing, wherein the final concentration of the standard oxalic acid solution is as follows: 1mM (90 mg), 1.5mM (135 mg), 2mM (180 mg), 3mM (270 mg), and 4mM (360 mg);
preparing a detection sample: 2-3mL of urine is taken, filtered for 2 times by a 0.22 mu m sterile syringe filter for injection, 950 mu L +50 mu L of oxalic acid standard solution with the concentration of 40mM is taken and added into a glass test tube, and the mixture is fully and evenly mixed;
(2) Adding 1mL of 100mM calcium chloride solution into a standard sample and a detection sample test tube, uniformly mixing, and then placing in a 37 ℃ incubator for standing reaction for 1 h;
(3) Taking out the test tube, centrifuging at 4000rpm for 15min by using a centrifuge, removing the supernatant, collecting crystals, washing the crystals once by using phosphate buffer (TBS), centrifuging at 4000rpm for 10min by using the centrifuge again, removing the supernatant, drying the crystals, and dissolving the crystals by using anhydrous hydrochloric acid;
(4) Detecting absorbance of the dissolved solution by a spectrophotometer at the wavelength of 215 nm;
(5) The OD of the standard solution was used to calculate a standard curve, the amount of oxalic acid was expressed in mg/L, and the urine was first expressed by the formula: OD (urine) = OD (test sample) - OD (1 mM oxalic acid) Obtaining the OD value of the urine, and the urinary calculus forming coefficient (UCI) = OD (urine) Standard curve coefficient, results are expressed in mg/L;
(6) The urinary calculus formation factor is multiplied by the urine volume in 24 hours, and is shown as mg/day.
The urine calculus forming coefficient can reflect the balance state of calcium ion-related formation Dan Yinzi (mainly oxalic acid) in urine, not only can reflect the saturation state of urine, but also can evaluate the risk of urinary calculus formation, and has certain auxiliary effects on early screening and postoperative follow-up of urinary calculus.
The beneficial technical effects of the invention are as follows:
(1) The core of the kit is that calcium oxalate in urine is activated to form in a limited time and at a limited temperature (in an incubator at 37 ℃) by adding a certain amount of oxalic acid and a sufficient amount of calcium chloride. And (3) fully dissolving the crystals after collecting the crystals, detecting the absorbance of the solution at OD215nm by using a spectrophotometer, and calculating the stone forming coefficient of the urine through a standard curve.
(2) The detection method has the advantages that the whole chemical reaction process is non-toxic and harmless, the reaction product has no residue and no pollution, and the reagent required by the detection does not contain any fluorescent agent; the spectrophotometer and the incubator are conventional laboratory instruments, special laboratory instruments are not needed, the experiment steps are simple, the operation is easy, batch detection can be realized, and the spectrophotometer and the incubator are suitable for clinical and laboratory popularization.
(3) The prior related methods for detecting the urine saturation aim to describe the amount of oxalic acid required at the beginning of the formation of crystals in the urine, and consequently, the whole saturation and the equilibrium state of the urine cannot be expressed. The method can reflect the whole balance state of the lithogenous ions and the lithogenous ions in the urine, takes the lithogenous coefficient as a result, and can visually reflect the lithogenous state and risk of the urine sample.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flow chart of the detection method of the kit of the present invention;
FIG. 2 is a standard curve of UCI values at different wavelengths;
fig. 3 UCI value standard curve (OD 215) test one;
FIG. 4 UCI value Standard Curve (OD 215) test two;
fig. 5 UCI value standard curve (OD 215) test three;
FIG. 6 ROC analysis (UCI concentration);
FIG. 7ROC analysis (UCI content).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The urinary calculus stone forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with the concentration of 40mM, oxalic acid solution with the concentration of 160mM and calcium chloride solution with the concentration of 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Example 2
The urinary system calculus stone becomes stone coefficient detect reagent box, contain STD reagent and Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 60mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Example 3
The urinary calculus stone forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 80mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter of 20mm diameter.
Example 4
The urinary calculus stone forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 120mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Example 5
The urinary system calculus stone becomes stone coefficient detect reagent box, contain STD reagent and Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 60mM, oxalic acid solution with concentration of 120mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter of 20mm diameter.
Example 6
The urinary calculus stone forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 60mM, oxalic acid solution with concentration of 80mM, oxalic acid solution with concentration of 120mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Detection method example 1
The detection steps are shown in fig. 1, and the specific operations are as follows:
(1) Preparing standard substance and test sample
Preparing a standard substance: adding 950 mu L of deionized water and 50 mu L of standard oxalic acid solution with different concentrations into a glass test tube, and fully and uniformly mixing, wherein the final concentration of the standard oxalic acid solution is as follows: 1mM (90 mg), 1.5mM (135 mg), 2mM (180 mg), 3mM (270 mg), and 4mM (360 mg);
adding 1mL of 100mM calcium chloride solution into each standard test tube, uniformly mixing, and then placing the mixture into an incubator at 37 ℃ for standing reaction to obtain 1 h;
taking out the test tube, centrifuging for 15min at 4000rpm by using a centrifuge, removing supernatant, drying crystals, and dissolving the crystals by using anhydrous hydrochloric acid;
detecting absorbance (OD value) of the solution at 215nm with a spectrophotometer;
the OD values of the standard solutions were used to calculate a standard curve, which was expressed in mg/L.
(2) As shown in fig. 2: the detection of the absorbance of crystals under different wavelengths and a standard curve show that the UCI value can achieve the best effect at OD 215.
(3) As shown in fig. 3-5: the UCI value standard curve is repeatedly measured, and the standard curve is relatively stable at OD215, and R2 is more than 0.99.
Detection method example 2
(1) A sample detection method;
preparing a detection sample: 2-3mL of urine is taken, filtered for 2 times by a 0.22 mu m sterile syringe filter for injection, 950 mu L plus 50 mu L of 40mM oxalic acid standard solution is added into a glass test tube and is fully and evenly mixed;
adding 1mL of 100mM calcium chloride solution into a test sample tube, uniformly mixing, and then placing the mixture into a 37 ℃ incubator for standing reaction for 1 h;
taking out the test tube, centrifuging for 15min at 4000rpm by using a centrifuge, removing supernatant collecting crystals, washing the crystals once by using TBS buffer solution, centrifuging for 10min at 4000rpm by using the centrifuge again, removing supernatant, drying the crystals, and dissolving the crystals by using anhydrous hydrochloric acid;
detecting absorbance of the dissolved solution by a spectrophotometer at the wavelength of 215 nm;
urine first passes the formula: OD (urine) = OD (test sample) - OD (1 mM oxalic acid) Obtaining the OD value of the urine;
urinary calculus formation coefficient (UCI) = OD (urine) Standard curve factor x 24 hours urine volume, results are expressed in mg/day;
(2) Clinical testing
And randomly selecting 200 healthy people and 200 calculi patients, and detecting the calculus forming coefficient by using urine of the healthy people and the calculi patients. After baseline data (age, sex) were balanced, 71 (healthy population) and 72 (stone patients) were included, respectively, and the results (table 1) suggest that there was a significant difference in UCI values (concentration and content) between healthy population and stone patients.
Table 1: clinical trial test data
As shown in the results of ROC analysis of FIGS. 6-7, the method can well distinguish the urine of healthy people and calculus patients, and the AUC values are both more than 90%. Wherein the method achieves the clinical standard if the content is used as an index, the AUC value and 95% CI both exceed 90%.
The segmentation value (Cut Off value) of UCI detection is calculated from the result of ROC analysis. As shown in table 2: at a UCI of 165 mg/day, sensitivity was 83.33%, specificity was 97.18%, and accuracy was 90.21%. Therefore, the detection method and the detection result of the kit both meet the clinical use standard.
TABLE 2 UCI partitioning value calculation
Detection method example 3
As an example of one case:
(1) The patient was first ordered to collect 24 hours of urine, after which the total amount of 24 hours of urine was measured and 10 ml was left after a little mixing.
(2) 1ml of urine was taken with a 2 ml syringe and filtered twice with a 0.22 μm sterile syringe needle filter.
(3) Adding 950 μ L of the filtered urine into a test tube, adding 50 μ L of 40mM oxalic acid standard solution (STD kit), and mixing to obtain a detection sample.
(4) 1ml of 100mM calcium chloride solution (STD kit) was added to the test sample tube, mixed well, and then allowed to stand in an incubator (37 ℃ C.) for 1 hour.
(5) Taking out the test tube, centrifuging at 4000rpm for 15min by using a centrifuge, removing the supernatant, collecting the crystals, washing the crystals once by using TBS buffer solution, centrifuging at 4000rpm for 10min by using the centrifuge again, removing the supernatant, drying the crystals, and dissolving the crystals by using anhydrous hydrochloric acid.
(6) The dissolved solution was measured for absorbance (OD value) at 215nm using a spectrophotometer, and the result of this measurement was 1.17.
(7) OD value of urine = OD value of test substance-OD value of 1mM standard substance, and OD value of this urine is 1.17-0.54=0.63, as calculated using the standard curve of FIG. 4
(8) Urinary calculus formation coefficient (UCI value) = OD (urine) The standard curve factor x 90mg/L x 24 h urine volume (L/day), the results are shown as content mg/day. The urine has a UCI value =0.63/0.006 × 2.23 (L/day) =234.15 mg/day, which is greater than the partition value 165 mg/day, so the urine has a higher risk of calculus formation.
Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover in the claims the invention as defined in the appended claims.
Claims (9)
1. The urinary calculus stone forming coefficient detection kit is characterized by comprising:
a standard reagent comprising the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 160mM and calcium chloride solution;
a test article reagent comprising the following components: buffer pH 7.4 and 0.22 μm sterile needle filter.
2. The kit of claim 1, wherein: the standard reagent further comprises: oxalic acid solution with concentration of 60mM, oxalic acid solution with concentration of 80mM or/and oxalic acid solution with concentration of 120 mM.
3. The kit according to claim 1, wherein the concentration of the calcium chloride solution is 100mM/L.
4. The kit according to claim 1, wherein the buffer has a concentration of 1M/L and the filter has a diameter of 20mm.
5. The kit of claim 4, wherein the buffer is phosphate buffered saline.
6. Use of a kit according to any one of claims 1 to 5 for the detection of the urinary calculus stone formation coefficient.
7. The use of the kit according to claim 6 for detecting the urinary calculus stone forming coefficient, wherein the steps comprise:
(1) Preparing a standard substance and a detection sample;
(2) Adding 1mL of 100mM calcium chloride solution into a standard sample test tube and a detection sample test tube, uniformly mixing, and then placing in a 37 ℃ incubator for standing reaction to obtain 1 h;
(3) Taking out the test tube, centrifuging for 15min at 4000rpm by using a centrifuge, removing supernatant, collecting crystals, washing the crystals once by using a phosphate buffer, centrifuging for 10min at 4000rpm by using the centrifuge again, removing supernatant, drying the crystals, and dissolving the crystals by using anhydrous hydrochloric acid;
(4) Detecting absorbance (OD value) of the dissolved solution at 215nm with a spectrophotometer;
(5) OD values of the standard solutions were used to calculate a standard curve, the amount of oxalic acid was expressed in mg/L, and urine was first expressed by the formula: OD (urine) = OD (test sample) - OD (1 mM oxalic acid) Obtaining the OD value of the urine, and the urinary calculus forming coefficient (UCI) = OD (urine) Standard curve coefficient, results are expressed in mg/L;
(6) The stone formation factor of urine × 24-hour urine volume is shown in mg/day.
8. The application of the kit according to claim 7 in the detection of the urinary calculus stone forming coefficient is characterized in that in the step (1), the standard substances are: adding 950 mu L of deionized water and 50 mu L of standard oxalic acid solution with different concentrations into a glass test tube, and fully and uniformly mixing, wherein the final concentration of the standard oxalic acid solution is as follows: 1mM, 1.5mM, 2mM, 3mM and 4mM.
9. The application of the kit according to claim 7 in the detection of the urinary calculus stone forming coefficient, wherein in the step (1), the detection sample is prepared by: 2-3mL of urine was collected, filtered 2 times through a 0.22 μm sterile syringe filter for injection, and 950 μ L +50 μ L of a 40mM oxalic acid standard solution was added to a glass test tube and mixed well.
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