Specific embodiment
Embodiment of the present invention is described in detail in conjunction with the embodiments, it will be appreciated by those skilled in the art that
The following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.Actual conditions are not specified in embodiment
Person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, being can be with
Conventional products that are commercially available.
The present invention relates to a kind of kits of bleeding risk after detection thrombolysis, including serum creatinine detection reagent and leucocyte
Count reagent.
According to an aspect of the present invention, the invention further relates to serum creatinine detection reagents and white blood cell count(WBC) reagent to prepare
Application after detection thrombolysis in the kit of bleeding risk.
Preferably, as described above to apply, bleeding is bleeding after ST sections of elevation thrombolysis in myocardial infarction after the thrombolysis.
Current main creatinine assay method have chemical assay (alkaline picrate method), enzyme process, high performance liquid chromatography (HPLC),
Raman scattering method, isotope dilution mass spectrometry, capillary electrophoresis and electrode method etc..Isotope dilution mass spectrometry, Raman scattering
Method, high performance liquid chromatography (HPLC) are not suitable for the analysis of high-volume clinical samples, generally only as the reference method of creatinine assay, institute
Generally this method is not used to measure with clinical;Although capillary electrophoresis setting-out line range is wide, operation is relatively simple, needs to use
Special installation and the pretreatment for carrying out serum specimen, routine clinical use are more difficult;Alkaline picrate method and enzymatic assays creatinine
It is widely applied by clinic.Due to the interference of false creatinine, people have carried out alkaline picrate method various alkaline picrate method
It improves, to improve the authenticity of measurement result, and realizes automated analysis, become clinically used detection method, but its line
Property range and anti-interference ability are still not as good as enzyme process.Enzyme process kit interference resistance is stronger than picric acid method, and sample dosage is few, pollution
It is few, it is suitable for automatic clinical chemistry analyzer.Thus preferred, the serum creatinine detection reagent is enzyme process serum creatinine detection reagent.
Zymetology method is that have the characteristics that relatively stable and easily operated in thrombolysis bleeding detection of the invention.
Preferably, the kit of bleeding risk or application, the serum creatinine detection reagent after detection thrombolysis as described above
Including creatine kinase, kreatinase, sarcosine oxidase and peroxidase.
By screening, the present invention measures creatinine level using zymetology method.Creatinine generates flesh under sarcosine oxidase effect
Propylhomoserin, sarcosine generate hydrogen peroxide under sarcosine oxidase effect, and the latter pacifies under hydrogen peroxide enzyme effect with 4- amino
Colour generation product aubergine quinone imines is generated for than woods, ESPAS reaction, causes absorbance to increase at 545nm, passes through monitoring
Absorbance value rises to measure the concentration of creatinine at 545nm.
Preferably, the kit of bleeding risk or application, the serum creatinine detection reagent after detection thrombolysis as described above
Further include one of reaction buffer, color developing agent, cleaning agent, preservative or a variety of:
Preferably, the reaction buffer is trihydroxy methyl amino buffer, Goods buffer, glycine-NaOH buffering
Liquid, N-2- hydroxyethyl piperazine-N'-2- ethanesulfonic acid buffer, N- tri- (methylol) methylamino -2- hydroxy-propanesulfonic acid buffer, N-
Bis- (2- hydroxyethanesulfonic acid) buffers of three (methylol) methyl-2-amino ethanesulfonic acid buffers, piperazine-N, N-, 3- morpholine -2-
Hydroxypropionate sodium buffer, 3- (N- morpholine) ethanesulfonic acid sodium buffer, 4- (2- ethoxy) piperazine -1-2- hydroxy-propanesulfonic acid
Bis- (2- ethoxy) amino -2- hydroxy-propanesulfonic acid bufferings of buffer, N- (2- ethoxy) piperazine-N'-4- fourth sulfonate buffer, 3-
Liquid, 3- (ring amine) -2- hydroxyl -1- propane sulfonic acid buffer, 4- (2- ethoxy) -1- piperazine propane sulfonic acid buffer, (ring is by 3-
Amine) -1- propane sulfonic acid buffer, 3- N-morpholinyl buffer, N- tri- (methylol) methyl-3-aminopropanesulfonicacid acid buffer one
Kind is several;
Preferably, the color developing agent is N- ethyl-N- (hydroxyl -3- sulfopropyl) meta-aminotoluene, potassium ferrocyanide and 4-
Amino-antipyrine;
Preferably, the preservative is potassium sorbate, in sodium benzoate, sodium nitrite, Proclin series preservative
The specific substance of one of a kind of specific substance or paraben esters;
It is furthermore preferred that the Proclin series preservative is Proclin300;
It is furthermore preferred that the paraben esters are methyl p-hydroxybenzoate, ethyl-para-hydroxybenzoate, para hydroxybenzene first
One of propyl propionate, butyl p-hydroxybenzoate, p-Hydroxybenzoic acid isopropyl ester, p-Hydroxybenzoic acid isobutyl ester.
Preferably, serum creatinine detection reagent of the present invention is divided into the first reagent and the second reagent;
First reagent includes creatine kinase, kreatinase;
Second reagent includes sarcosine oxidase, peroxidase, N- ethyl-N- (hydroxyl -3- sulfopropyl) meta-aminotoluene
(TOOS), 4-AA (PAP).
Serum creatinine detection reagent detection sample detected can be serum or heparin blood plasma.
The stability of serum, blood plasma: 4~25 DEG C of preservations can stablize 3 days.
Wherein, measuring method principle are as follows:
Absorption of the orchil of generation in 545nm is directly proportional to creatine concentration in sample.
Analysis type: end-point method deducts reagent blank;
Incubation time: 5 minutes;
Reaction time: 5 minutes;
First reagent/sample/the second ratio of reagents: 1800/60/600.Specifically used method is as shown in figure 3, absorbance becomes
The calculation formula of change are as follows:
Δ A=[(A2-A1) calibrates quality control or sample cell]-[(A2-A1) blank tube].
The calculation method of concentration are as follows:
C sample=(Δ A sample/Δ A calibration) × C calibration;
C sample: for the concentration of specimens of measurement;
C standard: for the concentration of calibration object.
In addition, one or more detection reagents or tool can be combined for examining to further increase the accuracy of detection
The diagnosis of bleeding risk after thought-read vascular diseases, especially thrombolysis.
Current white blood cell count(WBC), which is detected, can be divided into capacitive, photoelectric type, laser class and electrical impedance according to principle difference
Type.Electrical impedance method detection leucocyte is invented by the Kurt (W.H.Coulter) in the U.S. earliest, is improved by decades, should
Testing principle is verified repeatedly in clinical practice.
By screening, present invention preferably employs electrical impedance method white blood cell count(WBC) reagent, having is Kurt theory of electrical impedance
Detect white blood cell count(WBC).According to Fig.4, after quantitative blood is drawn and is diluted by quantitative conducting solution, just
It is sent to sensing chamber.There is a small opening in each sensing chamber, is called detection aperture.Have in the two sides of detection aperture logical
There is the positive and negative electrode of constant-current dc electricity.When diluted haemocyte passes through when acting through detection aperture of constant negative pressure, electricity
D.C. resistance between pole will change.This resistance will form a kind of pulse that same blood cell volume size is proportional change
Change.The distribution of particles that these data about pulse change being collected into can be used to one, picture reflection size of blood cells is bent
Line.
The pulse of each cell is distributed according to its volume size and is stored in corresponding when carrying out cell analysis by detector
Volume channel in, the data that each channel is collected are counted relative number, are indicated in Y-axis, volume data is with ascend to heaven (fl)
For unit, indicate in X-axis.It can be 256 channels, each channel 1.64fl, foundation from 30~450fl points by leucocyte volume
Volume size is placed it in respectively in different channels, obtains the volume distributing histogram of leukocyte (as shown in Figure 5).
Preferably, the kit of bleeding risk or application, the white blood cell count(WBC) reagent after detection thrombolysis as described above
Ingredient include potassium dihydrogen phosphate, disodium hydrogen phosphate, cetyl trimethylammonium bromide, disodium ethylene diamine tetraacetate, ammonium oxalate
And glacial acetic acid.
It is furthermore preferred that the formula of the white blood cell count(WBC) reagent are as follows:
Potassium dihydrogen phosphate: 0.256~0.296g/L, disodium hydrogen phosphate: 9.05~9.45g/L, disodium ethylene diamine tetraacetate:
15~25g/L, cetyl trimethylammonium bromide: 7~8g/L, ammonium oxalate: 15~25g/L, glacial acetic acid: 20~30ml/L, it is molten
Agent is water.
Preferably, the kit of bleeding risk or application, the white blood cell count(WBC) reagent after detection thrombolysis as described above
It further include anti-coagulants;
Preferably, the anti-coagulants includes edta salt, heparinate, citrate, oxalates, hirudin, potassium fluoride, fluorination
One of sodium is a variety of;
It is furthermore preferred that the edta salt is specially the potassium, sodium or lithium salts of EDTA;
It is furthermore preferred that the heparinate is specially the sodium, lithium or ammonium salt of heparin;
It is furthermore preferred that the citrate is specially the sodium or sylvite of citric acid;
It is furthermore preferred that the oxalates is specially the sodium or sylvite of oxalic acid.
In some embodiments, white blood cell count(WBC) can carry out in the following way:
1) reagent.Reagent includes diluent ingredient and hemolytic agent ingredient, and configuration method: first matching buffer, in a graduated cylinder,
Claim 4.6 grams of potassium dihydrogen phosphate, add 500 milliliters of distilled water, pH value 5.5, take 30 milliliters it is spare.In another graduated cylinder, claim phosphoric acid hydrogen
9.5 grams of disodium, add 1000 milliliters of distilled water, after dissolution, take out 30 milliliters and do not have to, pH value is controlled 8.5, by two graduated cylinder solution
Mixing, pH value 8.5 claim 7.5 grams of cetyl trimethylammonium bromide, 20 grams of disodium ethylene diamine tetraacetate, 20 grams of ammonium oxalate, will be upper
It states buffer and adds to 1000 milliliters, 25 milliliters of ice acetic acid again after heating for dissolving are filtered, filtrate pH value 5.5 to 5.8 with filter paper
Between.The reagent of pre-configuration can be used for diluted blood cell, prevents blood cell aggregation and adhesion, is used for haemolysis red blood cell, beats
Broken red blood cell, in favor of white blood cell count(WBC).
2) sample requirement: venous blood collection is anticoagulant using EDTA-K2.Capillary blood sampling should wipe First Blood, so as not to it is mixed
Entering tissue fluid influences result.Collection of specimens is completed to measure after placing 5-10 minutes.
3) operating procedure:
A. small test tube 1 adds pre-configured leucocyte dilution+hemolytic agent 0.38ml.
B. 20 μ l of peripheral blood is accurately drawn with micropipet, wipe outside tip more than blood, will suction pipe be inserted into it is dilute in small test tube
The bottom for releasing liquid, gently releases blood, and draws supernatant liquor, and cleaning suction pipe is secondary, mixes.
C. pond is filled, 2~3min is stood, passes through respective aperture to leucocyte.
D. white blood cell count(WBC) is carried out with Counting software.
4) it calculates: it is quantitative using the time, it by the liquid volume in the unit time is also constant when negative pressure is constant
, deviation that may be present is corrected using the counting for being carried out continuously same time twice, the result that this is counted twice compared to pair,
It is quoted within the acceptable range as a result, carrying out third time counting if beyond normal acceptable range.Utilize software pair
Histogram analyze judging.
According to an aspect of the present invention, the invention further relates to bleeding wind after a kind of ST sections of elevation thrombolysis in myocardial infarction of prediction
The method of danger, comprising:
For meeting the patient of thromboembolism treatment indication, obtained in patient's measuring samples using kit measurement as described above
Serum creatinine is horizontal and white blood cell count(WBC) data, transfer patient and symptom occur to consultation time, systolic pressure value and diabetes
History whether there is or not data, above-mentioned all data are distinguished into assignment and calculate gross score, by the gross score with pre-establish it is total
The corresponding relationship of bleeding risk is compared to obtain bleeding risk knot after thrombolysis after-ST sections of elevation thrombolysis in myocardial infarction of score
Fruit.
Preferably, the measuring samples include whole blood, serum or blood plasma.
Preferably, method as described above, by the method for above-mentioned all data difference assignment are as follows:
The following institute of corresponding relationship of bleeding risk after-ST sections of elevation thrombolysis in myocardial infarction of the gross score pre-established
Show:
According to an aspect of the present invention, the invention further relates to a kind of forecasting systems of bleeding risk after thrombolysis, such as Fig. 1 institute
Show, the system comprises:
Admission controller, serum creatinine detection part, the first assignment module, white blood cell count(WBC) component, the second assignment module, sample
This information receiving module, third assignment module and Risk Calculation module and reporting system main interface;
The admission controller is for assessing whether detected object meets thromboembolism treatment indication, will be by inspection pair if meeting
The sample to be examined of elephant is passed to serum creatinine detection part and the white blood cell count(WBC) component, and starts the sample information and receive function
It can module;
The serum creatinine that the serum creatinine detection part is used to detect in the sample to be examined is horizontal, and serum creatinine is detected and is tied
Fruit is passed to the first assignment module and carries out assignment;
The white blood cell count(WBC) component is for will carry out white blood cell count(WBC) in the sample to be examined, and by white blood cell count(WBC) knot
Fruit is passed to the second assignment module and carries out assignment;
The sample information receiving module for receive detected object information and be passed to the third assignment module into
Row assignment;
The Risk Calculation module receives the first assignment module, the second assignment module and the third assignment
The assignment of module, and be calculated bleeding risk after thrombolysis according to the total score of each assignment;
The reporting system main interface is for receiving the sample information receiving module and/or the Risk Calculation mould
The information of block, and bleeding risk assessment result after thrombolysis is exported.
Wherein, thromboembolism treatment indication can be judged according to standard well known in the art, such as: heart of Europe disease in 2017
Learn to provide in the ST-Elevation Acute Myocardial Infarction practice guidelines formulated: if in diagnosis ST-Elevation Acute Myocardial Infarction
It cannot row emergency PCI in time in 2 hours afterwards, it is proposed that the patient of no clear contraindication is after symptom appearance
Thromboembolism treatment is carried out in 12 hours.
The serum creatinine detection part can be examined for serum creatinine detection reagent as described above and this field routine serum creatinine
Survey pertinent instruments (especially enzyme process serum creatinine detects pertinent instruments).
The white blood cell count(WBC) component can be white blood cell count(WBC) reagent as described above and this field conventional white cytometer
Number pertinent instruments (especially electrical impedance method white blood cell count(WBC) pertinent instruments).
The system when in use, only can only be shown in client for serum creatinine detection part described in typing, described white
The interface of the output result of cell count component, and the interface also supports that being manually entered sample information receiving module is connect
The information (as shown in Figure 2) of receipts, and other component and/or module are hidden in backstage.After inputting information, click " vertical
Calculate " button, it can enter information into the Risk Calculation module, the main boundary of reporting system is then directly displayed at client
Face.
Preferably, after thrombolysis as described above bleeding risk forecasting system, controlled if the detected object does not meet thrombolysis
Indication is treated, then information is directly exported the reporting system main interface and generates suggestion intervention or drug is controlled by the admission controller
The report for the treatment of, while the system is closed by the admission controller.
Preferably, after thrombolysis as described above bleeding risk forecasting system, the first assignment module passes through with lower section
Formula carries out assignment:
If 0 μm of ol/L≤88.4 μm of serum creatinine < ol/L, is scored at 0;
If 88.4 μm of ol/L≤176.8 μm of serum creatinine < ol/L, are scored at 5;
If serum creatinine >=176.8 are scored at 9.
Preferably, after thrombolysis as described above bleeding risk forecasting system, the second assignment module passes through with lower section
Formula carries out assignment:
If white blood cell count(WBC)≤10 × 109, then it is scored at 0;
If white blood cell count(WBC) > 10 × 109, then it is scored at 4.
Preferably, after thrombolysis as described above bleeding risk forecasting system, the detected object information further includes being examined
, gender, weight, nationality, eating habit, life style, medication history, history of disease, there is symptom to medical at the age in the photo of object
One in time, household heredity factors, religious belief, heart condition, the Smoking And Drinking frequency, type of sports and the frequency, systolic pressure
Or it is multinomial;Preferably, the history of disease is diabetic history;
Preferably, the detected object information includes at least systolic pressure, diabetic history and symptom occurs to consultation time.
Wherein, in the present invention, systolic pressure, diabetic history and occur symptom to consultation time definition be art technology
Well known to personnel, such as:
1) blood pressure measurement: a. patient takes clinostatism or seat, 5-10 minutes tranquil, and bilateral ancon is placed in heart level;B. it is
Patient's turn-up cuff reveals arm (ancon stretches, and palmar is upward);C. sphygmomanometer is placed, is made at mercury " 0 " scale and at arteria brachialis, heart
In same level position, mercury cell switch is opened, drives residual air in sphygmomanometer girding to the greatest extent;D. it twines cuff is smooth in upper arm
Portion, lower edge are advisable away from fossa cubitalis 2-3CM, elastic referred to insertion one;E. arteria brachialis is touched, stethoscope is worn, stethoscope head is put in the upper arm
At arteriopalmus;F. valve screw-cap is closed, homogenous charge to auscultation brachial dance disappears, then increases 20-30mmHg.Slowly put
Gas, speed 4mmHg/s, head-up reading hear that the beating of the first sound is systolic pressure, and beating is changed voice/disappeared as diastolic pressure.
2) diabetes medical history confirms: diabetes of previously clarifying a diagnosis or the person that receives diabetes drug treatment;After being admitted to hospital on an empty stomach
Blood glucose >=7.0mmol/L (126mg/dl) or random plasma glucose >=11.1mmol/L (200mg/dl).
3) there is symptom to consultation time to confirm: symptom occur and be defined as before being this time admitted to hospital last breaking-out pectoralgia, uncomfortable in chest, dizzy
It faints, consultation time is defined as the assessment time started, and chronomere is accurate to hour.
Preferably, after thrombolysis as described above bleeding risk forecasting system, the third assignment module passes through with lower section
Formula carries out assignment:
If systolic pressure < 90mmHg, is scored at 0;
If 90mmHg≤systolic pressure < 140mmHg, is scored at 3;
If 140mmHg≤systolic pressure < 180mmHg, is scored at 6;
If aglycosuria medical history, is scored at 0;
If there is diabetic history, it is scored at 4;
If 0h≤consultation time < 3h, is scored at 0;
If 3h≤consultation time < 6h, is scored at 2;
If consultation time >=6h, is scored at 4.
Preferably, after thrombolysis as described above bleeding risk forecasting system, the Risk Calculation module is according to each assignment
Total score when be calculated bleeding risk after thrombolysis, the corresponding relationship of bleeding risk after the total score and the thrombolysis
Are as follows:
Preferably, after thrombolysis as described above bleeding risk forecasting system, the system also includes system personnel permissions
Control module, control content include:
A) access right of each functional component of and/or module controls;
B) typing, audit, printing, the personnel's permission differentiation for cancelling audit;
C) defines artificial screen locking or locks screen automatically function without operation.
Preferably, after thrombolysis as described above bleeding risk forecasting system, the letter that the reporting system main interface is shown
Breath include following content in one, it is multinomial or whole:
1) calls the detected object information in the specimen information receiving module and is shown;
2) record of date information and modification function;
The date information includes: the sampling time, the sample presentation time, the instrument detection date, user information date of entry, receives
Instrumental results date, audit report date, printed report date and send reporting day interim one or more.
3) calls bleeding risk evaluation text template after thrombolysis to show, and provides modification authority;
4) printing and establish customized report template that is reported;Custom item includes detected object number, report
Head, detected value, reference value, report picture, Health & Fitness Tip, auditor, printing people.
Preferably, after thrombolysis as described above bleeding risk forecasting system, bleeding risk is specially ST after the thrombolysis
Bleeding risk after section elevation thrombolysis in myocardial infarction.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations;To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced;And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.