CN107101933A - A kind of blood analysis composition and its application - Google Patents
A kind of blood analysis composition and its application Download PDFInfo
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- CN107101933A CN107101933A CN201710555692.XA CN201710555692A CN107101933A CN 107101933 A CN107101933 A CN 107101933A CN 201710555692 A CN201710555692 A CN 201710555692A CN 107101933 A CN107101933 A CN 107101933A
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- 238000004159 blood analysis Methods 0.000 title claims abstract description 34
- 239000000203 mixture Substances 0.000 title claims abstract description 33
- 239000003228 hemolysin Substances 0.000 claims abstract description 69
- 108010006464 Hemolysin Proteins Proteins 0.000 claims abstract description 68
- 206010018910 Haemolysis Diseases 0.000 claims abstract description 19
- 230000008588 hemolysis Effects 0.000 claims abstract description 19
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 26
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 22
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 13
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 13
- 235000011152 sodium sulphate Nutrition 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 12
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 11
- 235000019253 formic acid Nutrition 0.000 claims description 11
- 239000003381 stabilizer Substances 0.000 claims description 9
- 150000003839 salts Chemical group 0.000 claims description 5
- 239000000834 fixative Substances 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 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 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims 1
- 238000011109 contamination Methods 0.000 abstract description 21
- 238000002474 experimental method Methods 0.000 abstract description 11
- 210000003743 erythrocyte Anatomy 0.000 abstract description 10
- 238000004458 analytical method Methods 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 230000002949 hemolytic effect Effects 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 description 36
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 23
- 239000012153 distilled water Substances 0.000 description 17
- 238000000034 method Methods 0.000 description 17
- 102000012153 HLA-B27 Antigen Human genes 0.000 description 16
- 108010061486 HLA-B27 Antigen Proteins 0.000 description 16
- 229930040373 Paraformaldehyde Natural products 0.000 description 14
- 229920002866 paraformaldehyde Polymers 0.000 description 14
- 230000010165 autogamy Effects 0.000 description 13
- 235000002639 sodium chloride Nutrition 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 238000012360 testing method Methods 0.000 description 11
- 239000012530 fluid Substances 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 230000009514 concussion Effects 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 6
- 229910001868 water Inorganic materials 0.000 description 6
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 5
- 239000003513 alkali Substances 0.000 description 5
- 239000001099 ammonium carbonate Substances 0.000 description 5
- 235000012501 ammonium carbonate Nutrition 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 102000001554 Hemoglobins Human genes 0.000 description 4
- 108010054147 Hemoglobins Proteins 0.000 description 4
- IXCSERBJSXMMFS-UHFFFAOYSA-N hcl hcl Chemical compound Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 4
- 238000011017 operating method Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102100039856 Histone H1.1 Human genes 0.000 description 2
- 102100027368 Histone H1.3 Human genes 0.000 description 2
- 101001035402 Homo sapiens Histone H1.1 Proteins 0.000 description 2
- 101001009450 Homo sapiens Histone H1.3 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108010056995 Perforin Proteins 0.000 description 2
- 102000004503 Perforin Human genes 0.000 description 2
- 238000000149 argon plasma sintering Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000003219 hemolytic agent Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 101000606032 Pomacea maculata Perivitellin-2 31 kDa subunit Proteins 0.000 description 1
- 101000606027 Pomacea maculata Perivitellin-2 67 kDa subunit Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
Landscapes
- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to blood analysis field, more particularly to a kind of blood analysis composition and its application.The blood analysis composition lysed erythrocyte speed is fast, with low cost.The hemolysin strict control time provided with the present invention carries out hemolytic experiment, experiment analysis results and completely the same (Clinical Sensitivity and specificity are 100% with commercially available hemolysin experimental result, correctness is consistent, residual contamination rate result≤1%, the hemolysis of instrument residual contamination rate≤1%) is good, meets requirement of experiment.
Description
Technical field
The present invention relates to blood analysis field, more particularly to a kind of blood analysis composition and its application.
Background technology
Flow cytometry (flowcytometry FCM) is to carry out fast quantification point to individual cells or other biological particulate
Analysis and a special kind of skill of sorting.In analysis or assorting room, surround the individual cells or particulate treated in working fluid
By the light source of focusing, electric signal is produced, these signals represent the parameters such as light scattering, fluorescence, cell or particulate are determined with this
Physics and chemical property, and can sub-elect the cell subsets of high-purity according to these properties, with to its further culture or
Analysis.The liquid stream of wrapping cell is referred to as sheath fluid.Instrument is referred to as flow cytometer (flowcytometer FCM).Streaming is thin
Born of the same parents' art combines the multi-door subject such as optics, electronics, hydrodynamics, cytochemistry, immunology, laser technology and computer science
And technology, with the features such as detection speed is fast, accurate, measurement index is more, gathered data amount is big, analysis is comprehensive, method is flexible.Stream
Formula cell art has become one of common assay method in laboratory as a kind of quantitative analysis tech.
Blood analyser reagent mainly includes hemolysin, dye liquor.Hemolysin is mainly acidophilus hemolysin and basophilic hemolysin.
Blood analyser reagent is the strong surfactant of a class, and an effect is lysed erythrocyte, discharges hemoglobin (HGB),
Stable haemoglobin dervative is formed, the measure of hemoglobin is carried out.Its another aspect is lysed erythrocyte, is carried out white thin
Born of the same parents classify with counting.
Hemolysin (hemolysin/haemolysin), also known as cytolysin (cy-totoxin).Specifically it is thin
A part in cell lysis element, what cytolysin referred to bacterial secretory can make cytolytic toxin.Hemolysin belongs to perforation poison
Plain (pore-forming toxin), also known as attack membrane toxin (Mem-brane disrupting type), refer to it is any can make it is red
Cell dissolves and discharges the material of hemoglobin.
The effect of hemolysin and principle：
1st, hemolysin is tested for the examination of flow cytometry HLA-B27 antigens, and it is to dissolve the red blood cell in whole blood that it, which is acted on,.
HLA-B27 is I type human major histocompatibility's antigens, and the lymphocytic cell surface in leucocyte is mainly expressed in peripheral blood.
Anti- HLA-B27 is marked to be combined with lymphocyte with fluorescein, you can to use flow cytomery.
2nd, lysed erythrocyte：In favor of carrying out accurate fluorescein mark to leucocyte.It is red in flow cytometry tests
Cell volume and leucocyte volume have a region of coincidence, and the number of red blood cell is nearly 1,000 times of leucocyte number, influence
To the fluorescence antibody of significant cell (leucocyte) mark and sort, thus be expert at flow cytometry when, it is necessary to add haemolysis
Element, destroys red blood cell, could carry out fluorescein mark to leucocyte exactly.
Conventional hemolysin cost is about 1400 yuan every bottle at present, and scientific research cost is higher；Haemolysis needs about 10~15min,
Hemolysis time is long.
The content of the invention
In view of this, the present invention provides a kind of blood analysis composition and its application.The present invention is to do streaming conventional project
The examination of HLA-B27 antigens provides one kind hemolytic agent and its collocation method, and the hemolysin lysed erythrocyte speed is fast, and cost is low
It is honest and clean.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of blood analysis composition, including formic acid, surfactant and stabilizer.
In some specific embodiments of the present invention, stabilizer described in the blood analysis composition is salt.
In some specific embodiments of the present invention, the pH value of the blood analysis composition is 7.4.
In some specific embodiments of the present invention, salt described in the blood analysis composition is selected from sodium carbonate, chlorine
Change sodium or sodium sulphate.
In some specific embodiments of the present invention, the blood analysis composition also includes fixative.
In some specific embodiments of the present invention, the blood analysis composition also includes buffer solution and/or pH value
Conditioning agent.
In some specific embodiments of the present invention, in terms of mL/g/g, surface described in the blood analysis composition
Activating agent, the mass ratio of the stabilizer and the fixative are：(1~2):(1~2):(0.5~1).
In some specific embodiments of the present invention, sodium carbonate in stabilizer described in the blood analysis composition,
The mass ratio of sodium chloride and sodium sulphate is：1:(2~5):(5~10).
Present invention also offers application of the described blood analysis composition in haemolysis.
Present invention also offers a kind of hemolysin, including described blood analysis composition.
In some specific embodiments of the present invention, the hemolysin includes tri- components of A, B, C：
Component A is made up of formic acid and water, and the volume ratio of formic acid and water is 1:(800~1000).Preferably, formic acid and water
Volume ratio be 1:800.
B component is made up of Yan Heshui；Salt is made up of ammonium carbonate, sodium chloride and sodium sulphate；In terms of g/g/g/mL, ammonium carbonate,
Mass/mass/mass/volume of sodium chloride and sodium sulphate and water is 1:(2~5):(5~10):(150~200).As excellent
Choosing, in terms of g/g/g/mL, mass/mass/mass/volume of ammonium carbonate, sodium chloride and sodium sulphate and water is 1:2:5:170.
Component C is made up of paraformaldehyde and PBS, in terms of g/mL, the quality volume of paraformaldehyde and PBS
Than for 1:(100~150).Preferably, in terms of g/mL, the mass volume ratio of paraformaldehyde and PBS is 1:100.
In some specific embodiments of the present invention, the volume ratio of tri- components of A, B, C is (1~2):(1~
2):(0.5~1).Preferably, the volume ratio of tri- components of A, B, C is 1:1:0.5.
Present invention also offers the compound method of hemolysin.Comprise the following steps：
A liquid：In terms of mL/g, the volume mass ratio of formic acid and distilled water is 1：(800~1000)；Room temperature preservation；
B liquid：Ammonium carbonate, sodium chloride and sodium sulphate composition；In terms of g/g/g/mL, ammonium carbonate, sodium chloride and sodium sulphate and water
Mass/mass/mass/volume be 1:(2~5):(5~10):(150~200)；
C liquid：The mass ratio of paraformaldehyde and buffer solution is 1:(100~150)；
PH value regulator (such as 1mol/L sodium hydroxide) is added, pH value meta-alkali is sufficiently stirred for hydrotropy solution；
PH value is adjusted to 7.4 with pH value regulator (1mol/L hydrochloric acid) again.
The operating procedure of hemolysin：First plus A liquid, concussion observation adds B liquid, after concussion immediately to bright (about 5~10s)
Add C liquid.A liquid：B liquid：The volume ratio of C liquid is (1~2):(1~2):(0.5~1).
In some specific embodiments of the present invention, the compound method of the hemolysin is as follows：
A liquid：Formic acid 0.6mL+500mL distilled water；Room temperature preservation.
B liquid：Sodium carbonate (Na2CO3)：3g、
Sodium chloride：7.25g、
Sodium sulphate：15.65g；
It is dissolved in 300mL distilled water；500mL is complemented to after being completely dissolved with distilled water again.
C liquid：Paraformaldehyde 3g is added in 300mL sheath fluids (PBS)；
Sodium hydroxide (NaOH) is added, pH value meta-alkali is sufficiently stirred for hydrotropy solution,
500mL is complemented to after paraformaldehyde is completely dissolved with sheath fluid again.
First plus A liquid 1.0mL, concussion observation adds B liquid 2mL immediately to bright (about 5~10s), and C liquid is added after concussion
0.5mL。
PH value is adjusted to 7.4 with 1mol/L hydrochloric acid (HCL) again.
In some specific embodiments of the present invention, the preferred distilled water of water, distilled water or ultra-pure water.
Present invention also offers a kind of kit, including described blood analysis composition or described hemolysin.
The present invention provides a kind of blood analysis composition and its application.The blood analysis composition includes formic acid, surface and lived
Property agent and stabilizer, provide hemolytic agent and its collocation method, hemolysin dissolving a kind of to be streaming conventional project HLA-B27
Red blood cell speed is fast, with low cost.
The blood analysis composition that the present invention is provided has the following advantages that：
1st, cost savings：
Autogamy hemolysin cost：Calculated according to the specification of autogamy hemolysin, every bottle of solid articles raw material can enough configure hemolysin
33 times, configurable haemolysis pixel volume is 500mL every time, and it is 33*500=16500mL that configuration hemolysin volume is available for altogether.Autogamy
Hemolysin price often covers about 100 yuan or so of (paraformaldehyde, sodium carbonate etc. whole raw materials).
Producer's hemolysin cost：Hemolysin specification：100mL, by 1:It is used to test after 9 ratio distilled water diluting, i.e.,
Every bottle can be configured to hemolysin solvent for 1000mL.Every bottle of producer's hemolysin price is about 1400 yuan.It is converted into autogamy hemolysin
Every bottle of configurable volume 16500mL, cost is about 16500/1000*1400=23100 members.
To sum up, if converted to the hemolysin of autogamy, isometric producer's finished product hemolysin cost be autogamy hemolysin into
This 23100/100=231 times.
2nd, saving of time：
The commercialization hemolysin provided with producer, the hemolysis time is 15 minutes, and is only with the autogamy hemolysin hemolysis time
10 seconds, shorten the stand-by period of experiment and the stand-by period of instrument.
3rd, the present invention has done control experiment to hemolysin and commercially available hemolysin for B27 project clinical samples.From B27 projects
Sample in each 20 of the samples of negative and positive two levels, the hemolysin strict control time provided with the present invention are provided
Carry out hemolytic experiment, experiment analysis results and completely the same (Clinical Sensitivity and specificity are equal with commercially available hemolysin experimental result
For 100%, correctness is consistent, residual contamination rate result≤1%, and the hemolysis of instrument residual contamination rate≤1%) is good, meets
Requirement of experiment.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described.
Fig. 1 shows H1-1 residual contamination rate result 6347；
Fig. 2 shows H1-3 residual contamination rate result 6102；
Fig. 3 shows L1-1 residual contamination rate result 0；
Fig. 4 shows L1-3 residual contamination rate result 0；
Fig. 5 shows H2-1 residual contamination rate result 6581；
Fig. 6 shows H2-3 residual contamination rate result 6332；
Fig. 7 shows H2-1 residual contamination rate result 1；
Fig. 8 shows H2-3 residual contamination rate result 0；
Fig. 9 shows H3-1 residual contamination rate result 5968；
Figure 10 shows H3-3 residual contamination rate result 6270；
Figure 11 shows H3-1 residual contamination rate result 3；
Figure 12 shows H3-3 residual contamination rate result 0.
Embodiment
The invention discloses a kind of blood analysis composition and its application, those skilled in the art can be used for reference in herein
Hold, be suitably modified technological parameter realization.In particular, all similar replacements and change are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.The method of the present invention and application are by preferably implementing
Example is described, related personnel substantially can not departing from present invention, in spirit and scope to method described herein and
Using being modified or suitably changing with combining, to realize and apply the technology of the present invention.
Raw materials used and reagent can be bought by market in blood analysis composition that the present invention is provided and its application.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1
Collocation method is as follows：
A liquid：Formic acid 0.6mL+500mL distilled water；Room temperature preservation.
B liquid：Sodium carbonate (Na2CO3)：3g、
Sodium chloride：7.25g、
Sodium sulphate：15.65g；
It is dissolved in 300mL distilled water；500mL is complemented to after being completely dissolved with distilled water again.
C liquid：Paraformaldehyde 3g is added in 300mL sheath fluids (PBS)；
Sodium hydroxide (NaOH) is added, pH value meta-alkali is sufficiently stirred for hydrotropy solution,
500mL is complemented to after paraformaldehyde is completely dissolved with sheath fluid again.
PH value is adjusted to 7.4 with 1mol/L hydrochloric acid (HCL) again.
Hemolysin operating procedure：
Test tube tilts addition A liquid 1.0mL and vibrated to completely bright (3~5s of strict control duration of oscillation)：Dissolution time mistake
Short, not exclusively, light scattering resolution ratio is reduced hematoclasis, has red blood cell contamination in lymphocyte door；Dissolution time is long, broken
Piece increase, setting of the interference Instrument to T lymphocytes door.
B liquid 1mL vibrations are added immediately.
It is subsequently added C liquid 0.5mL.
Note：(it is recommended that when specimen amount is big, some processes of haemolysis point are carried out, every 3 pipe is a process, a process definition
For：Terminate from A liquid is added to C liquid is added).
Embodiment 2
Collocation method is as follows：
A liquid：Formic acid 0.6mL+480mL distilled water；Room temperature preservation.
B liquid：Sodium carbonate (Na2CO3)：3g、
Sodium chloride：15g、
Sodium sulphate：15g；
It is dissolved in 300mL distilled water；500mL is complemented to after being completely dissolved with distilled water again.
C liquid：Paraformaldehyde 5g is added in 750mL sheath fluids (PBS)；
Sodium hydroxide (NaOH) is added, pH value meta-alkali is sufficiently stirred for hydrotropy solution,
500mL is complemented to after paraformaldehyde is completely dissolved with sheath fluid again.
PH value is adjusted to 7.4 with 1mol/L hydrochloric acid (HCL) again.
Hemolysin operating procedure：
First plus A liquid 2mL, concussion observation adds B liquid 1.5mL immediately to bright (about 5~10s), and C liquid is added after concussion
1mL。
Embodiment 3
Collocation method is as follows：
A liquid：Formic acid 0.6mL+600mL distilled water；Room temperature preservation.
B liquid：Sodium carbonate (Na2CO3)：3g、
Sodium chloride：6g、
Sodium sulphate：30g；
It is dissolved in 300mL distilled water；500mL is complemented to after being completely dissolved with distilled water again.
C liquid：Paraformaldehyde 5g is added in 750mL sheath fluids (PBS)；
Sodium hydroxide (NaOH) is added, pH value meta-alkali is sufficiently stirred for hydrotropy solution,
500mL is complemented to after paraformaldehyde is completely dissolved with sheath fluid again.
PH value is adjusted to 7.4 with 1mol/L hydrochloric acid (HCL) again.
Hemolysin operating procedure：
First plus A liquid 1mL, concussion observation adds B liquid 2mL immediately to bright (about 5~10s), and C liquid is added after concussion
0.75mL。
The HLA-B27 antigens examination (flow cytometry) of embodiment 4 aids in hemolysin reagent comparison data
First, detecting system information：
Project：HLA-B27 antigens examination (flow cytometry)
Instrument title：BECKMAN COULTER FC500 flow cytometers
INSTRUMENT MODEL：Cytomics FC500MPL(SN:AV08034)
Reagent and manufacturer：HLA-B27 KIT BD companies
HLA-B27 BD FACS BD companies
Detection method：Flow cytometry
Control group：BD FACS finished product hemolysins；
Experimental group：The hemolysin that the embodiment of the present invention 1~3 is provided.
2nd, checking content and result
Correctness：
HLA-B27 has carried out BD FACS with Beckman Coulter FC500MPL (sequence number AT08034) instrument
The hemolysin result detection contrast that finished product hemolysin result and the embodiment of the present invention 1~3 are provided, the result of analysis is consistent, therefore
HLA-B27 experiments have passed through correctness checking.It is shown in Table 2.
Specificity：
HLA-B27 has carried out BDFACS finished product haemolysis with Cytomics FC500MPL (sequence number AT08034) instrument
The hemolysin result detection contrast that plain result and the embodiment of the present invention 1~3 are provided, the result of analysis is consistent, therefore HLA-B27 is tested
Specificity is 100%.It is shown in Table 1.
Sensitivity：
It is molten that HLA-B27 has carried out BD FACS finished products with Cytomics FC500MPL (sequence number AT08034) instrument
The hemolysin result detection contrast that sanguinin result and the embodiment of the present invention 1~3 are provided, the result of analysis is consistent, therefore HLA-B27 is real
It is 100% to test sensitivity.It is shown in Table 1.
Residual contamination rate：
Tested using machine on standard microballoon, follow-on test 3 times, calculate the granule number in demarcation region, be calculated as respectively
H1-1, H1-2, H1-3, carry out after a sample cell recoil circulation, then enter the test of line blank quantity, follow-on test 3 times calculates demarcation
The granule number in region, is calculated as L respectively1-1, L1-2, L1-3；Circulated 3 times by this program, residual contamination rate is calculated according still further to below equation
(Ci) maximum, is taken.
Ci=(L1-1-L1-3)/(H1-1-H1-3) * 100%
Criterion：Ci≤1%
Testing result：
As shown in Fig. 1~Figure 12.
H1-1：6347；H1-3：6102；L1-1：0；L1-3：0；C1=0%.
H2-1：6581；H2-3：6332；L2-1：1；L2-3：0；C2=0.04%.
H3-1：5968；H3-3：6270；L3-1：3；L3-3：0；C3=0.1%.
Before control experiment, to instrument carry out 3 times detection residual contamination rate result≤1%, instrument residual contamination rate≤
1%.Meet the requirements.
Detection time is in May, 2015.
The clinical specificity of table 1 and susceptible clinical degree assess table
The correctness of table 2 evaluates proof list (different detecting systems)
Summary result, hemolysin HLA-B27 detection projects correctness that the present invention is provided, specificity, sensitivity, takes
Band pollution rate the result is subjected to, available for clinical samples detection.
Embodiment 5
Control group：BD FACS finished product hemolysins；
Experimental group：The hemolysin that the embodiment of the present invention 1~3 is provided.
Production, test operation situation are shown in Table 3.
1st, cost savings：
(1) autogamy hemolysin cost：Calculated according to the specification of autogamy hemolysin, every bottle of solid articles raw material can enough configure haemolysis
Element 33 times, configurable haemolysis pixel volume is 500mL every time, and it is 33*500=16500mL that configuration hemolysin volume is available for altogether.From
Often cover with hemolysin price about 100 yuan or so of (whole such as paraformaldehyde, sodium carbonate raw materials).
(2) producer's hemolysin cost：Hemolysin specification：100mL, by 1:It is used to test after 9 ratio distilled water diluting,
I.e. every bottle can be configured to hemolysin solvent for 1000mL.Every bottle of producer's hemolysin price is about 1400 yuan.It is converted into autogamy haemolysis
Every bottle of configurable volume 16500mL of element, cost is about 16500/1000*1400=23100 members.
(3) to sum up, if converted to the hemolysin of autogamy, isometric producer's finished product hemolysin cost is the hemolysin of autogamy
23100/100=231 times of cost.
Table 3
| Group | Control group | Experimental group |
| Production cost (member) | 23100 | 100# |
| Hemolysis time | 15min | 10#s |
*Show has significant difference (P ＜ 0.05) compared with control group,#Show has significant difference (P ＜ compared with control group
0.01)。
2nd, saving of time：
The commercialization hemolysin provided with producer, the hemolysis time is 15min, and is only with the autogamy hemolysin hemolysis time
10s, shortens the stand-by period of experiment and the stand-by period of instrument.
Embodiment 6
Control group 1：Component and compound method be the same as Example 1~3, pH value is 7.2.
Control group 2：Component and compound method be the same as Example 1~3, pH value is 7.3.
Control group 3：Component and compound method be the same as Example 1~3, pH value is 7.5.
Control group 4：Component and compound method be the same as Example 1~3, pH value is 7.6.
Experimental group：Embodiment 1~3, pH value is 7.4.
Comparative result is shown in Table 4.
Table 4
As shown in Table 4, the pH value of hemolysin be 7.4 when, the hemolysis time is shorter, and hemolyzing effect is more complete, cell fragment compared with
Few, upper machine testing effect is preferable, with preferable sensitivity, specificity and correctness.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of blood analysis composition, it is characterised in that including formic acid, surfactant and stabilizer.
2. blood analysis composition according to claim 1, it is characterised in that its pH value is 7.4.
3. blood analysis composition according to claim 1 or 2, it is characterised in that the stabilizer is salt；The salt choosing
From sodium carbonate, sodium chloride or sodium sulphate.
4. the blood analysis composition according to any one of claims 1 to 3, it is characterised in that also including fixative.
5. the blood analysis composition according to any one of Claims 1-4, it is characterised in that also including buffer solution and/or
PH value regulator.
6. the blood analysis composition according to claim 4 or 5, it is characterised in that in terms of mL/g/g, the surface-active
Agent, volume/mass/quality of the stabilizer and the fixative are：(1~2):(1~2):(0.5~1).
7. the blood analysis composition according to any one of claim 1 to 6, it is characterised in that carbonic acid in the stabilizer
The mass ratio of sodium, sodium chloride and sodium sulphate is：1:(2~5):(5~10).
8. application of the blood analysis composition in haemolysis according to any one of claim 1 to 7.
9. a kind of hemolysin, it is characterised in that including the blood analysis composition as described in any one of claim 1 to 7.
10. a kind of kit, it is characterised in that including the blood analysis composition as described in any one of claim 1 to 7 or such as
Hemolysin described in claim 9.
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| CN111239120A (en) * | 2020-02-26 | 2020-06-05 | 绍兴市人民医院 | Blood calcium ion detection test paper |
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| CN101246158A (en) * | 2008-03-21 | 2008-08-20 | 王亚东 | Hemolytic agent for measuring white blood cell in hemocyte |
| CN102226804A (en) * | 2011-03-28 | 2011-10-26 | 中国人民解放军总医院 | Hemolytic agent for blood leukocyte five-classification counting and application thereof |
| CN102662067A (en) * | 2012-05-20 | 2012-09-12 | 烟台卓越生物技术有限责任公司 | Cyanide-free hemolysin for determining hemoglobin concentration and carrying out white blood cell count by groups |
| CN103323582A (en) * | 2013-06-18 | 2013-09-25 | 南京普朗医疗设备有限公司 | Leukocyte classification hemolytic agent and kit thereof |
| WO2016145432A1 (en) * | 2015-03-12 | 2016-09-15 | Zoetis Services Llc | Pyolysin methods and compositions |
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|---|---|---|---|---|
| CN101246158A (en) * | 2008-03-21 | 2008-08-20 | 王亚东 | Hemolytic agent for measuring white blood cell in hemocyte |
| CN102226804A (en) * | 2011-03-28 | 2011-10-26 | 中国人民解放军总医院 | Hemolytic agent for blood leukocyte five-classification counting and application thereof |
| CN102662067A (en) * | 2012-05-20 | 2012-09-12 | 烟台卓越生物技术有限责任公司 | Cyanide-free hemolysin for determining hemoglobin concentration and carrying out white blood cell count by groups |
| CN103323582A (en) * | 2013-06-18 | 2013-09-25 | 南京普朗医疗设备有限公司 | Leukocyte classification hemolytic agent and kit thereof |
| WO2016145432A1 (en) * | 2015-03-12 | 2016-09-15 | Zoetis Services Llc | Pyolysin methods and compositions |
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