CN109321523A - A kind of blood additive - Google Patents
A kind of blood additive Download PDFInfo
- Publication number
- CN109321523A CN109321523A CN201811202136.5A CN201811202136A CN109321523A CN 109321523 A CN109321523 A CN 109321523A CN 201811202136 A CN201811202136 A CN 201811202136A CN 109321523 A CN109321523 A CN 109321523A
- Authority
- CN
- China
- Prior art keywords
- blood
- additive
- value
- nucleic acid
- heparin tube
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- 210000004369 blood Anatomy 0.000 title claims abstract description 81
- 239000008280 blood Substances 0.000 title claims abstract description 81
- 239000000654 additive Substances 0.000 title claims abstract description 61
- 230000000996 additive effect Effects 0.000 title claims description 57
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 31
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 31
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 31
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000003112 inhibitor Substances 0.000 claims abstract description 9
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 7
- 229940127219 anticoagulant drug Drugs 0.000 claims abstract description 7
- 239000000872 buffer Substances 0.000 claims abstract description 7
- 101710163270 Nuclease Proteins 0.000 claims abstract description 6
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 5
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- 231100000614 poison Toxicity 0.000 claims abstract description 5
- 230000006907 apoptotic process Effects 0.000 claims abstract description 4
- 239000011814 protection agent Substances 0.000 claims abstract description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 60
- 229960002897 heparin Drugs 0.000 claims description 60
- 229920000669 heparin Polymers 0.000 claims description 60
- 238000001514 detection method Methods 0.000 claims description 27
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 25
- 239000006228 supernatant Substances 0.000 claims description 24
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- 230000000694 effects Effects 0.000 claims description 9
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- OOBJCYKITXPCNS-REWPJTCUSA-N (3s)-5-(2,6-difluorophenoxy)-3-[[(2s)-3-methyl-2-(quinoline-2-carbonylamino)butanoyl]amino]-4-oxopentanoic acid Chemical compound O=C([C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)C=1N=C2C=CC=CC2=CC=1)C(C)C)COC1=C(F)C=CC=C1F OOBJCYKITXPCNS-REWPJTCUSA-N 0.000 claims description 5
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- MNQZXJOMYWMBOU-VKHMYHEASA-N D-glyceraldehyde Chemical compound OC[C@@H](O)C=O MNQZXJOMYWMBOU-VKHMYHEASA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
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- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 claims description 3
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- YJIYWYAMZFVECX-UHFFFAOYSA-N 2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]anilino]acetic acid acetyloxymethyl ester Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=CC=C1OCCOC1=CC=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O YJIYWYAMZFVECX-UHFFFAOYSA-N 0.000 claims description 2
- PBVAJRFEEOIAGW-UHFFFAOYSA-N 3-[bis(2-carboxyethyl)phosphanyl]propanoic acid;hydrochloride Chemical compound Cl.OC(=O)CCP(CCC(O)=O)CCC(O)=O PBVAJRFEEOIAGW-UHFFFAOYSA-N 0.000 claims description 2
- 239000004475 Arginine Substances 0.000 claims description 2
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 claims description 2
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 claims description 2
- 108010024636 Glutathione Proteins 0.000 claims description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
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- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 2
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- 235000010298 natamycin Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- QUBQYFYWUJJAAK-UHFFFAOYSA-N oxymethurea Chemical compound OCNC(=O)NCO QUBQYFYWUJJAAK-UHFFFAOYSA-N 0.000 description 1
- 229950005308 oxymethurea Drugs 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000010118 platelet activation Effects 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-M salicylate Chemical compound OC1=CC=CC=C1C([O-])=O YGSDEFSMJLZEOE-UHFFFAOYSA-M 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
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Abstract
The invention discloses a kind of blood additives, it includes anti-coagulants 6.0-8.8g/100ml；Nucleic acid inhibitor 2.0-8.4g/100ml；Cytotostatic agent 4-20g/100ml；Purine 0-0.31g/100ml；Metabolic poison 2-10g/100ml；Inhibitors of apoptosis 0.021-0.21g/100ml；Nuclease protection agent 0.1-1g.Non- Cl‑Buffer 90-110mM, pH 7.3-7.5.The present invention can be stored at room temperature blood 14 days, not influence to detect.
Description
Technical field
The present invention relates to a kind of blood additives.
Background technique
2015, liquid Biopsy was chose as one of annual ten quantum jump technologies, because containing tumour source in blood
Free nucleic acid and become the most important research object of liquid Biopsy.Effectively realize that the liquid biopsy based on blood depends on
Two key factors, first is that detection technique sensitive enough is needed, because of plasma DNA (cell-free DNA, cfDNA)
In, Circulating tumor DNA (circulating tumor DNA) proportion is very low, with the development of detection technique, at present
CtDNA detection sensitivity is up to 0.2%.Second, due to that can not be separated in first time after blood collection in real world
Blood plasma and the extraction and detection for carrying out ctDNA, therefore, effective preservation of blood are also most important.However, due to containing in blood
Nuclease abundant is easily degraded ctDNA, while a large amount of leucocytes contained in blood are easy hair in preservation or transportational process
Raw rupture, the genomic DNA (gDNA) thus released the ctDNA that further dilution is originally very rare, to detection structure
At stern challenge.In addition, blood, in preservation or transportational process, red blood cell is also easily broken generation haemolysis, what is released is each
Kind substance (such as hemoglobin) can also interfere with the detection of subsequent ctDNA.
Currently, the Preservation tactics and method to blood free nucleic acid have very much, but regrettably these are protected or are
Protect free nucleic acid or only protect leucocyte do not rupture or protective agent in contain to human body or the harmful ingredient of environment,
In short, still lack at this stage it is a kind of can comprehensively, the protection free nucleic acid of system, reduce the method that gDNA discharges.
Application No. is 201410534999.8 patents to disclose a kind of reagent of stable blood sample cell, can anti-hemostasis
Platelet is assembled and stablizes haemocyte at least 36 hours, but the patent is not directed to the information of free nucleic acid protection.
Application No. is 201611214323.6 patents to disclose the heparin tube of a kind of protection and stable dissociative DNA, protects
Protecting includes imidazolidinyl urea in agent component, and application No. is 201610778832.5 patents to disclose tire in a kind of maternal blood
The preservative agent of youngster's dissociative DNA and its vacuum blood collection tube of composition contain diazonium imidazolidinyl urea and/or formaldehyde, Shen in preservative agent
It number please disclose a kind of for protecting the blood anticoagulant of dissociative DNA for 201510132774.4 patent, which contains
Formaldehyde releaser, including 1,3- dihydroxymethyl -5,5- Dimethyl Hydan, sodium hydroxy methyl glycinate, soluble metyl hydroxybenzoate, nipalgin
At least one of ethyl ester sodium or soluble propylhydroxybenzoate, application No. is 201510088419.1 patent disclose one kind can be
The circulation dissociative DNA evacuated collection of blood sample is acquired and saved under normal temperature condition, and additive therein contains preservative, including 2-
Bromo- 2- nitropropane -1,3- glycol, bi-imidazolidinyl urea, quaternary ammonium salt, alcohol, sodium hydroxy methyl glycinate, hydantoins, formaldehyde
And its one or more of derivative, imidazolidinyl urea, dimethylol urea, oxazolidine or methanol, application publication number US2014/
The patent of 0080112A1 discloses a kind of heparin tube, and additive contains preservative, selected from imidazolidinyl urea and double imidazolidinyls
Urea.
It is to use formaldehyde or formaldehyde releaser that the above patent, which is used to solve one of strategy and method of free nucleic acid protection,
Though certain protection free nucleic acid can be played the role of and stablize cell, the crosslinking of the substances such as nucleic acid, albumen, serious shadow will lead to
Ring the extraction and detection of downstream nucleic acid.
Application No. is 201710380209.9 patents to use natural antiseptic agent (epsilon-polylysine, tea polyphenols, natamycin
One of) chemical preservative is replaced, solve the problems, such as toxicity, but then, multiple research (Chen XL and Yin
SM,Chin J Pathophysiol,2004；Taylor KA et al., J Platlets, 2017) show that Cl- ion can draw
Play platelet activation and aggregation.And the patent still continues to use the buffer system containing Cl- ion, is unfavorable for the stabilization of blood platelet, pushes away
Survey the stabilization that may be further unfavorable for red blood cell.Other are further included application number or are awarded using the patent of Cl- ion buffer system
Power number is 201510062267.8,201610778832.5,201510132774.4,201580042850.0 and US9376709
Deng.
In addition, 4 DEG C of cryo-conservations and transport are mostly used, however, the program has at this stage in order to realize the transport of blood
Clearly disadvantageous place, first even under 4 DEG C of low temperature, nuclease is still active, will lead to the degradation of free nucleic acid,
Secondary, 4 DEG C of transports increase cost burden, and finally and most important shortcoming is that cryogenic conditions are also easy activation blood platelet,
Stimulate haemolysis.
It can be seen that there is also clearly disadvantageous in the prior art.
Summary of the invention
The purpose of the present invention is to provide a kind of new blood additives, are stored at room temperature and transport for blood, to be used for
Subsequent detection.Blood additive of the invention, can not only effectively prevent plasma free nucleolysis, but can effective stabilized leukocyte,
GDNA/gRNA release is reduced, red blood cell can be also protected, the blood additive of haemolysis is effectively reduced.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of blood additive, every 100 milliliters of additives include following components
Anti-coagulants 6.0-8.8g (EDTAP dipotassium ethylene diamine tetraacetate, ethylenediamine tetra-acetic acid tripotassium, disodium ethylene diamine tetraacetate, Chinese holly
One or more of rafter acid sodium).Anti-coagulants can anti-hemostasis-coagulation, while also can inhibit DNA enzymatic and RNA enzyme.The preferred second of anti-coagulants
Ethylenediamine tetraacetic acid (EDTA) dipotassium.
(BAPTA-AM, Tris (2- carboxyethyl)-phosphine-hydrochloride, vanadyl ribonucleotide are multiple by nucleic acid inhibitor 2.0-8.4g
Close one or more of object, dithiothreitol (DTT), dithioerythritol, coke diethyl phthalate, ammonium sulfate, urea).Nuclease suppression
Preparation can inhibit with DNA enzymatic RNA enzyme, avoid the degradation of dissociative DNA and RNA in blood plasma.The preferred urea of nucleic acid inhibitor；
Cytotostatic agent 4-20g (polyethylene glycol, polyvinyl alcohol, adonitol, melezitose, Tween-80, cysteine,
One or more of reduced glutathione).It is preferred that polyvinyl alcohol 1788 (degree of polymerization 1700, alcoholysis degree 88%), polyethylene
Alcohol can be used as matrix and wrap up blood cell, stablize cellular morphology.
Purine 0-0.31g is the one or two of adenine, guanine, and the addition of purine can maintain red cell morphology,
It is not easy to deform and rupture.Preferably 0.11-0.31g.It is preferred that adenine.
Metabolic poison 2-10g (glyceraldehyde-3-phosphate, 1,3 diphospho glycerate acid, glyceraldehyde, sodium fluoride, phosphoenol
One or more of formula pyruvic acid).Metabolic poison metabolism capable of inhibiting cell reduces genomic nucleic acids in cellular process
Release.Preferably glycerine aldehyde；
Inhibitors of apoptosis 0.021-0.21g (one or both of Q-VD-OPh, Z-VAD-FMK), it is capable of inhibiting cell to wither
It dies, prevents the release of genomic nucleic acids.Q-VD-OPh and Z-VAD-FMK is commercially available to be obtained, for example, purchase is difficult to understand from Sigma
Delhi surprise company.It is preferred that Q-VD-OPh.
Nuclease protection agent 0.1-1g (glycine betaine, trehalose, glycine, arginine, spermine, spermidine), can protect nucleic acid,
Especially RNA prevents the degradation of nucleic acid.It is preferred that glycine, spermine；
Non- Cl- buffer 90-110mM, pH 7.3-7.5 (citric acid/sodium citrate buffer, phosphate buffer,
One of MOPS buffer), it can avoid activation blood platelet, while preventing haemolysis.It is preferred that MOPS buffer or phosphate-buffered
Liquid.It is preferred that 100mM, pH 7.4.
When in use, the volume ratio with blood sample is 1:30-40, preferably 1:35 to blood additive of the present invention.
The present invention also provides a kind of novel blood additive protecting effect evaluations of programme, include the following steps:
Firstly, (it is thin such as to possess the H1975 that EGFR T790M is mutated to the tumor cell line for possessing the mutation of some specific gene
Born of the same parents system or the H3122 cell line for possessing ALK fusion mutation) after progress regular growth culture 2-3 days, culture solution supernatant is drawn, from
The heart, (preferably 500g is centrifuged 10min) shift supernatant into new centrifuge tube；
Secondly, acquiring healthy volunteer respectively with control EDTA heparin tube using the heparin tube containing blood additive to be assessed
Peripheral blood, the above-mentioned culture solution supernatant of equal volume is added, carries out being transported at room temperature test later and is stored at room temperature test；
Again, free nucleic acid is extracted respectively, is detected using qPCR method, to evaluate the effect of additive.In present invention side
In method, by introducing mutated gene into healthy human blood, the variation of mutation Ct value is mainly used for indicating nucleolysis, and internal control
The variation of Ct value is used to indicate nucleolysis and leucocyte rupture, and the two combination can distinguish both effects, to precisely assess
The protecting effect of additive.
The detection of used PCR detection architecture is limited to 1%, therefore, prescribes a time limit not less than detection, the variation for being mutated Ct is main
Indicate that nucleolysis, nucleolysis will lead to mutation Ct value and increase, degradation amplitude is (2Folder becomes Δ Ct- 1) × 100%.
The variation of internal control Ct value is used to indicate nucleolysis and leucocyte rupture, and nucleolysis causes Ct value to increase, leucocyte
Rupture cause Ct value reduction, leucocyte level of breakage be 2 (Folder becomes Δ Ct+Internal control Δ Ct) -1 × 100%.
Beneficial effect
1, there is document report Cl- ion that blood platelet can be activated to lead to platelet aggregation, the present invention is according to blood platelet and red thin
Relationship between born of the same parents creatively connects Cl- ion and red blood cell rupture (haemolysis), therefore contrast test contains Cl-
Ion and buffer system without Cl- ion, achieve unexpected effect, the results showed that, it is slow without Cl- ion
It is strong (Fig. 1) to rush solution system anti-hemolysis ability.
2, be transported at room temperature 4 days, the present invention can better stable red blood cell, there is stronger anti-hemolysis ability (Fig. 2)
3, it is stored at room temperature 14 days and (during preservation, heparin tube slowly turns upside down 10 times daily, then 2mL is taken to be used for
Nucleic acid extraction and PCR detection), the present invention has stronger anti-hemolysis ability (Fig. 3)
4, it is transported at room temperature 4 days, the present invention can more effectively protect free nucleic acid (DNA and RNA) and blood leucocyte
5, the present invention can be stored at room temperature blood 14 days, not influence to detect.
6, the documents referred in background technique, the assessment to additive effect, the overwhelming majority are by comparing some
Gene or house-keeping gene change in the Ct value of two timing nodes (such as 0 day and T days) to carry out, however, influencing the variation of Ct value
Effect is on the contrary, therefore, only for two factors --- free nucleic acid degradation causes Ct value to become larger, and gDNA release causes Ct value to become smaller ---
The two factors can not be distinguished well by detecting the variation of gene C t value, can not accurately assess the true effect of additive
Fruit.Occasionally have by mixing the cell line dna extracted into blood and assessed, however, the DNA extracted is completely exposed
(albumen has been digested in extraction process) can not reflect the state of true dissociative DNA in blood, therefore can not accurate evaluation
Additive effect.One kind proposed by the present invention can system evaluation additive effect evaluation scheme, can both assess dissociative DNA or trip
Palliating degradation degree from RNA can also assess leucocyte level of breakage and gDNA/gRNA releasing degree.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 difference buffer system anti-hemolysis ability；
Anti-hemolysis compares after Fig. 2 is transported at room temperature 4 days.
(0,4,7,14 day) anti-hemolysis compares during Fig. 3 is stored at room temperature.
(0,4,7,14 day) Ct value variation diagram during Fig. 4 is stored at room temperature.
Specific embodiment
Each component in following embodiment is unless otherwise instructed to be commercially available.
Blood additive protecting effect evaluation of the present invention, includes the following steps:
Firstly, (it is thin such as to possess the H1975 that EGFR T790M is mutated to the tumor cell line for possessing the mutation of some specific gene
Born of the same parents system or the H3122 cell line for possessing ALK fusion mutation) after progress regular growth culture 2-3 days, culture solution supernatant is drawn, from
The heart, (preferably 500g is centrifuged 10min), shifting supernatant, (culture solution supernatant at this time contains only target cell point into new centrifuge tube
Free nucleic acid out is secreted, cell is free of)；
Secondly, acquiring healthy volunteer respectively with control EDTA heparin tube using the heparin tube containing blood additive to be assessed
Peripheral blood, the above-mentioned culture solution supernatant of equal volume is added, carries out being transported at room temperature test later and is stored at room temperature test；
Again, free nucleic acid is extracted respectively, and using qPCR method, (the qPCR reagent that the present invention uses is Xiamen Ai De biology
" human EGFR mutated gene detection kit (fluorescent PCR method) " and " mankind EML4- of medical sci-tech limited liability company production
ALK fusion gene detection kit (fluorescent PCR method) ") detection, to evaluate the effect of additive.In the methods of the invention, pass through
Mutated gene is introduced into healthy human blood, the variation of mutation Ct value is mainly used for indicating nucleolysis, and internal control Ct value changes
It is used to indicate nucleolysis and leucocyte rupture, the two combination can distinguish both effects, to precisely assess additive
Protecting effect.
The detection of used PCR detection architecture is limited to 1%, therefore, prescribes a time limit not less than detection, the variation for being mutated Ct is main
Indicate that nucleolysis, nucleolysis will lead to mutation Ct value and increase, degradation amplitude is (2Folder becomes Δ Ct- 1) × 100%.
The variation of internal control Ct value is used to indicate nucleolysis and leucocyte rupture, and nucleolysis causes Ct value to increase, leucocyte
Rupture leads to the reduction of Ct value, and leucocyte level of breakage is { 2(folder becomes Δ Ct+ internal control Δ Ct)- 1 } × 100%.
It should be noted that when calculating free nucleic acid degradation amplitude and leucocyte level of breakage, it is contemplated that used
The CV value (PCR intrinsic fluctuating error) of PCR detection architecture is within 5%, therefore, it is considered that, the variation of Ct value is no more than 0.5
It can be considered that Ct value does not change, just there is practical indicative significance more than 0.5.
Embodiment 1
Blood additive provided in this embodiment, every 100mL include following component (formula 1):
The present embodiment provides following additives containing Cl-, and as control, every 100mL includes following component:
According to above-mentioned formula, corresponding reagent is weighed respectively, is dissolved in 100mM phosphate buffer and is made into the 100mL present invention
Be formulated 1 additive, every heparin tube 285.7 μ L containing the additive prepares heparin tube containing Cl- in the same way, while with it is general
Logical EDTA heparin tube compares test.Specific step is as follows:
1, blood is carried out respectively with the heparin tube of EDTA heparin tube, the heparin tube of 1 additive containing formula and the additive containing Cl-
Acquisition, each tube blood sampling volume are 10mL, are slowly turned upside down immediately after blood sampling heparin tube 10 times, keep blood and additive sufficiently mixed
It is even；
2, it places and saves under normal temperature conditions, blood gentle inversion is mixed 10 after daily observation heparin tube haemolysis situation
It is secondary, transportation environment is simulated, after placing 5 days, heparin tube is subjected to 2000g and is centrifuged 10 minutes with separated plasma, observation, more every kind
Heparin tube haemolysis situation.
Experimental result shows (Fig. 1), and after being stored at room temperature 5 days, significant hemolysis, blood plasma occur for the heparin tube of the additive containing Cl-
In peony, moderate haemolysis occurs for EDTA heparin tube, and blood plasma is in light red, and the heparin tube containing additive of the present invention is insoluble
Blood, blood plasma are still in faint yellow, it is seen that can effectively prevent haemolysis using additive of the present invention, and haemolysis is most instead for additive containing Cl-
Seriously.
Embodiment 2:
Blood additive provided in this embodiment, every 100mL include following component (formula 2):
Test is compared according to above-mentioned formula additive preparation, while with common EDTA heparin tube.Specific step is as follows:
1, it is formulated the heparin tube of 1 additive with EDTA heparin tube, containing embodiment 1 and is formulated adopting for 2 additives containing embodiment 2
Blood vessel carries out blood collection respectively, and each tube blood sampling volume is 10mL, slowly turns upside down heparin tube 10 times immediately after blood sampling, makes blood
Liquid is mixed well with additive；
2, after transporting 4 days under normal temperature conditions, heparin tube is subjected to 2000g and is centrifuged 10 minutes with separated plasma, observation, ratio
More every kind of heparin tube haemolysis situation.
Experimental result shows (Fig. 2), and after being transported at room temperature 5 days, obvious haemolysis occurs for EDTA heparin tube, and contains inventive formulation
1 or be formulated 2 additives heparin tube obvious haemolysis does not occur, it is seen that using additive of the present invention can effectively stable red blood cell,
In transportational process haemolysis occurs for anti-Hemostatic Oral Liquid.
Embodiment 3:
Using 1 blood additive of embodiment (formula 1), using common EDTA heparin tube as control, carry out being transported at room temperature reality
It tests, the specific steps are as follows:
1, tumor cell line H1975 (EGFR T790M is mutated positive cell line), (is contained using 1640 culture medium of RMPI
10% fetal calf serum), after 37 DEG C of 5%CO2 are cultivated 3 days, culture solution supernatant is shifted into clean centrifuge tube；
2,2000g is centrifuged 10min, shifts in the new clean centrifuge tube of culture solution supernatant value；
3, using heparin tube and common EDTA heparin tube containing additive of the present invention, 3 volunteer's peripheral bloods are acquired respectively,
Every pipe acquires 10mL, slowly turns upside down immediately heparin tube 10 times；
4, step 2 culture solution supernatant is taken, is vortexed after mixing, 50 μ L is taken to be added in every pipe 10mL blood of step 3 respectively, is delayed
Slow reverse 10 mixings；
5, take 3mL blood into clean centrifuge tube from every heparin tube, as T0 sample, every heparin tube residue
Blood be transported at room temperature 4 days, equally respectively take 3mL blood into clean centrifuge tube again, as T4 sample；
6, the separation of T0 and T4 sample blood plasma: blood sample is put into a centrifuge, and 2000g is centrifuged 10min, careful to draw
Supernatant is into new centrifuge tube, then 10000g is centrifuged 10min, and careful 1mL supernatant of drawing is into new centrifuge tube, to obtain blood plasma；
7, plasma DNA is extracted, extracts kit selects the core of Xiamen Ai De biological medicine Science and Technology Co., Ltd.
Acid extracts reagent (model: Circulating DNA)；
8, PCR is detected: being detected using the human EGFR mutated gene of Xiamen Ai De biological medicine Science and Technology Co., Ltd.
Kit (fluorescent PCR method) carries out PCR detection to the plasma DNA of extraction, because H1975 cell line has EGFR gene 20
Exon T790M mutation, therefore detect T790M mutation.
Experimental result: entering to have organized 3 volunteers, and every volunteer acquires peripheral blood, every kind of heparin tube using 3 kinds of heparin tubes
Respectively at 0 day and transport and extracts dissociative DNA at 4 days separated plasmas, 18 DNA samples carry out PCR detection altogether, PCR result with
Ct value is presented, and each sample standard deviation has 2 Ct values: T790M is mutated Ct value and internal control Ct value, wherein in detection limit, T790M is prominent
It is related to dissociative DNA degradation to become the variation of Ct value, degradation will lead to the raising of Ct value, increases more multilist and shows that degradation is more serious；Internal control Ct
It is related to dissociative DNA degradation and leucocyte rupture (gDNA release) to be worth variation, but the former causes Ct value to increase, raising is got over multilist and shown
It degrades more serious, the latter causes Ct value to become smaller, and the more multilist that becomes smaller shows that leucocyte rupture is more serious.According to different heparin tube Ct values
Variation may compare heparin tube to the protecting effect of dissociative DNA and haemocyte.Testing result is as shown in table 1.
As it can be seen from table 1 T790M mutation Ct value of the present invention averagely increases 0.26 after being transported at room temperature 4 days, internal control Ct
Value has averagely become smaller 0.17, and compares EDTA heparin tube T790M mutation Ct value and increase 0.75, and internal control Ct value has become smaller 4.97,
Illustrate that the present invention can more effectively protect dissociative DNA, reduce its degradation, also can effectively protect leucocyte, reduces caused by its rupture
The release of gDNA；And EDTA heparin tube dialogue cytoprotective effect is very poor, rupture is serious during transportation, and gDNA is largely released
It puts, while dissociative DNA also has a degree of degradation.
Table 1 is transported at room temperature 4 days Ct value situations of change
Embodiment 4:
Using 1 blood additive of embodiment (formula 1), using common EDTA heparin tube as control, carry out being transported at room temperature reality
It tests, the specific steps are as follows:
1, tumor cell line H3122 (ALK merges positive cell line) (contains 10% tire ox blood using 1640 culture medium of RMPI
Clearly), after 37 DEG C of 5%CO2 are cultivated 3 days, culture solution supernatant is shifted into clean centrifuge tube；
2,2000g is centrifuged 10min, shifts in the new clean centrifuge tube of culture solution supernatant value；
3, using heparin tube and common EDTA heparin tube containing additive of the present invention, 3 volunteer's peripheral bloods are acquired respectively,
Every pipe acquires 10mL, slowly turns upside down immediately heparin tube 10 times；
4, step 2 culture solution supernatant is taken, is vortexed after mixing, 500 μ L is taken to be added in every pipe 10mL blood of step 3 respectively,
Slowly reverse 10 mixings；
5, take 3mL blood into clean centrifuge tube from every heparin tube, as T0 sample, every heparin tube residue
Blood be transported at room temperature 4 days, equally respectively take 3mL blood into clean centrifuge tube again, as T4 sample；
6, the separation of T0 and T4 sample blood plasma: blood sample is put into a centrifuge, and 2000g is centrifuged 10min, careful to draw
Supernatant is into new centrifuge tube, then 10000g is centrifuged 10min, and careful 1mL supernatant of drawing is into new centrifuge tube, to obtain blood plasma；
7, plasma free RNA is extracted, extracts kit selects the core of Xiamen Ai De biological medicine Science and Technology Co., Ltd.
Acid extracts reagent (model: Circulating DNA), and what which extracted is total nucleic acid, including DNA and RNA；
8, RT-PCR is detected: merging base using the mankind EML4-ALK of Xiamen Ai De biological medicine Science and Technology Co., Ltd.
Because plasma free RNA of the detection kit (fluorescent PCR method) to extraction carries out RT-PCR detection.
Experimental result: entering to have organized 3 volunteers, and every volunteer acquires peripheral blood, every kind of heparin tube using 3 kinds of heparin tubes
Respectively 0 day and 4 days separated plasmas of transport and the free RNA of extraction, 18 free RNA samples carry out RT-PCR detection altogether, tie
For fruit with the presentation of Ct value, the Ct value variation of ALK is related to RNA palliating degradation degree, and Ct value increases more multilist and shows more serious, the No Ct that degrades
Refer at the end of PCR sets amplification cycles number (36) do not there is signal rise yet, that is to say, that Ct > 36.According to different heparin tubes
The variation of Ct value may compare heparin tube to the protecting effect of free RNA.Testing result is as shown in table 2.
Table 2 is transported at room temperature 4 days Ct value situations of change
From table 2 it can be seen that compared with 0 day, ALK Ct value variation of the present invention is smaller, and right after blood is transported at room temperature 4 days
It is changed significantly according to EDTA heparin tube (without Ct value after transport 4 days), illustrates that the present invention can more effectively protect free RNA, reduce
It is degraded.
Embodiment 5:
Using 2 blood additive of embodiment (formula 2), using common EDTA heparin tube as control, carries out room temperature and stand guarantor
Deposit experiment, the specific steps are as follows:
1, tumor cell line H1975 (EGFR T790M is mutated positive cell line), (is contained using 1640 culture medium of RMPI
10% fetal calf serum), after 37 DEG C of 5%CO2 are cultivated 3 days, culture solution supernatant is shifted into clean centrifuge tube；
2,2000g is centrifuged 10min, shifts in the new clean centrifuge tube of culture solution supernatant value；
3, using the heparin tube containing additive of the present invention, the peripheral blood of 3 volunteers is acquired respectively, and every acquisition 2 is managed, often
Pipe acquires 10mL, slowly turns upside down immediately heparin tube 10 times；Above-mentioned 3 volunteers are taken a blood sample to EDTA with same method and are adopted
In blood vessel；
4, step 2 culture solution supernatant is taken, is vortexed after mixing, takes 100 μ L to be added respectively (same in the 20mL blood of step 3
Two pipe blood of the name same heparin tube of volunteer merge), slowly reverse 10 mixings, are then dispensed into 4 centrifuge tubes, often
Pipe 5mL is stored at room temperature preservation；
5,1 pipe separated plasma is taken respectively in 0 day (in 4 hours), 4 days, 7 days, 14 days 4 timing nodes, observe haemolysis
Situation extracts plasma DNA, finally carries out PCR detection, and step is same as Example 3.
Experimental result: haemolysis situation as shown in figure 3, PCR testing result as shown in table 3 and fig. 4.
Table 3 is stored at room temperature 14 days Ct value situations of change
It can be seen that room temperature from table 3 and Fig. 4 and stand preservation 14 days, the present invention can preferably protect dissociative DNA (T790M
Ct value averagely increases 0.38, and EDTA heparin tube increases 1.26) and protection leucocyte, reduction gDNA discharge (internal control Ct value
Averagely become smaller 0.40, and 5.44) EDTA heparin tube has become smaller.Therefore, the present invention can protect blood under the conditions of being stored at room temperature
Up to 14 days, and EDTA heparin tube then could.
Claims (10)
1. a kind of blood additive, it is characterised in that: include:
Anti-coagulants 6.0-8.8g/100ml, including EDTAP dipotassium ethylene diamine tetraacetate, ethylenediamine tetra-acetic acid tripotassium, ethylenediamine tetra-acetic acid two
At least one of sodium, sodium citrate；
Nucleic acid inhibitor 2.0-8.4g/100ml, including BAPTA-AM, Tris (2- carboxyethyl)-phosphine-hydrochloride, vanadyl ribose
At least one of nucleosides compound, dithiothreitol (DTT), dithioerythritol, coke diethyl phthalate, ammonium sulfate, urea；
Cytotostatic agent 4-20g/100ml, including polyethylene glycol, polyvinyl alcohol, adonitol, melezitose, Tween-80, half
At least one of cystine, reduced glutathione；
Purine 0-0.31g/100ml, at least one including adenine, guanine；
Metabolic poison 2-10g/100ml, including glyceraldehyde-3-phosphate, 1,3- diphosphoglyceric acid, glyceraldehyde, sodium fluoride, phosphorus
At least one of sour enol pyruvic acid；
At least one of inhibitors of apoptosis 0.021-0.21g/100ml, including Q-VD-OPh, Z-VAD-FMK；
At least one of nuclease protection agent 0.1-1g, including glycine betaine, trehalose, glycine, arginine, spermine, spermidine；
Non- Cl- buffer 90-110mM, pH 7.3-7.5, including citric acid/sodium citrate buffer, phosphate buffer,
At least one of MOPS buffer.
2. a kind of blood additive according to claim 1, it is characterised in that: purine concentration is 0.11-0.31g/
100ml。
3. a kind of blood additive according to claim 1, it is characterised in that: purine is adenine.
4. a kind of blood additive according to claim 1, it is characterised in that: anti-coagulants is EDTAP dipotassium ethylene diamine tetraacetate；
Nucleic acid inhibitor is urea；Cytotostatic agent is polyvinyl alcohol 1788；Metabolic poison is glyceraldehyde.
5. a kind of blood additive according to claim 1, it is characterised in that: inhibitors of apoptosis Q-VD-OPh；Nucleic acid
Protective agent is glycine or spermine.
6. a kind of blood additive according to claim 1, it is characterised in that: non-Cl-Buffer is MOPS buffer or phosphorus
Phthalate buffer.
7. as blood additive as claimed in any one of claims 1 to 6 application method, for by the blood additive according to blood
The volume ratio of liquid sample is that the ratio of 1:30-40 is added in blood sample.
8. the method for being used for evaluating blood additive protecting effect, includes the following steps:
1) firstly, drawing culture after carrying out regular growth culture 2-3 days to the tumor cell line for possessing the mutation of some specific gene
Liquid supernatant, centrifugation so that culture solution supernatant is contained only the free nucleic acid that target cell secrets out of, be free of cell, transfer supernatant to it is new from
In heart pipe；
2) periphery of healthy volunteer is acquired respectively with control EDTA heparin tube using the heparin tube containing blood additive to be evaluated
The above-mentioned culture solution supernatant of equal volume is added in blood, carries out being transported at room temperature test later and is stored at room temperature test；
3) free nucleic acid is extracted respectively, is detected using qPCR method, to evaluate the effect of additive, by into healthy human blood
Mutated gene is introduced, the variation of mutation Ct value is mainly used for indicating nucleolysis, and the variation of internal control Ct value is used to indicate nucleic acid drop
Solution and leucocyte rupture, the two combination can distinguish both effects, to obtain the assessment result of additive protecting effect.
9. being used for the method for evaluating blood additive protecting effect as claimed in claim 8, which is characterized in that blood to be evaluated
Additive is blood additive as claimed in any one of claims 1 to 6.
10. being used for the method for evaluating blood additive protecting effect as claimed in claim 8, which is characterized in that be not less than
When detection limit 1%, it is mutated the variation instruction nucleolysis of Ct, nucleolysis will lead to mutation Ct value and increase, and degradation amplitude is
The variation of internal control Ct value is used to indicate nucleolysis and leucocyte rupture, and nucleolysis causes Ct value to increase, leucocyte rupture
Lead to the reduction of Ct value, leucocyte level of breakage is
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| CN111096756A (en) * | 2019-12-23 | 2020-05-05 | 湖北金杏科技发展有限公司 | Blood collection tube for medical clinical examination and preparation method thereof |
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| CN108893524A (en) * | 2018-06-04 | 2018-11-27 | 北京启衡星生物科技有限公司 | The protective agent of dissociative DNA in blood plasma |
| CN110432914A (en) * | 2019-07-26 | 2019-11-12 | 宁儿医院股份有限公司 | Sequencing type is anti-to obscure vacuum blood collection tube |
| CN111096756A (en) * | 2019-12-23 | 2020-05-05 | 湖北金杏科技发展有限公司 | Blood collection tube for medical clinical examination and preparation method thereof |
| CN111057704A (en) * | 2019-12-31 | 2020-04-24 | 广州市金圻睿生物科技有限责任公司 | Dewaxing cracking combination liquid, kit and method for extracting DNA from paraffin section sample |
| CN111057704B (en) * | 2019-12-31 | 2022-07-05 | 广州市金圻睿生物科技有限责任公司 | Dewaxing cracking combination liquid, kit and method for extracting DNA from paraffin section sample |
| CN111423975A (en) * | 2020-03-23 | 2020-07-17 | 重庆医科大学附属永川医院 | Vacuum sampling tube capable of inactivating viruses and preparation method thereof |
| CN111500704A (en) * | 2020-04-28 | 2020-08-07 | 广州市金圻睿生物科技有限责任公司 | Human fetus chromosome aneuploidy detection kit and method |
| CN111500704B (en) * | 2020-04-28 | 2023-10-27 | 广州市金圻睿生物科技有限责任公司 | Kit and method for detecting human fetal chromosome aneuploidy |
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Application publication date: 20190212 |