CN101672853A - Blood cell analyzer calibrator and preparation process thereof - Google Patents
Blood cell analyzer calibrator and preparation process thereof Download PDFInfo
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- CN101672853A CN101672853A CN 200910186118 CN200910186118A CN101672853A CN 101672853 A CN101672853 A CN 101672853A CN 200910186118 CN200910186118 CN 200910186118 CN 200910186118 A CN200910186118 A CN 200910186118A CN 101672853 A CN101672853 A CN 101672853A
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Abstract
The invention uses synergetic combination effect of multiple cytoactive protection solution and a stabilizer, and a protective agent provides nutrition components and living environment required by maintaining normal physiological activity for blood cells for anaerobic glycolysis; the stabilizer containing cytolemma and cytoplasm fixed substances with lower concentration can slowly permeate the cytolemma to act on the cytoplasm and firmly combine with biomolecule of cell components such as protein, enzyme, lipid and the like effectively, thus enabling white blood cells, red blood cells and blood platelets to be fixed in different degrees and get effective protection and enabling blood cells to keep apparent morphological characteristics thereof. The blood cells in protection and good stabilized treatment is subjected to assignment according to traceability link delivery procedures and reference methods thereof, and can be used as blood cell analyzer calibrators for clinic usage. The invention uses stem complete blood cell (CBC) from human beings or animals, thus having good stability; the invention can be used in microscope manual counting and cell staining analysis, and has longerexpirydate compared with the current full blood cell calibrators.
Description
Technical field
The present invention relates to the preparation and cellanalyzer calibration object (thing) fabricating technology thereof of bioactivity-protected liquid of haemocyte and stabilizing agent.
Background technology
In the cellanalyzer quality control process, the calibration or the correction of instrument are absolutely necessary, and new instrument is installed or all should be carried out the calibration of instrument after the maintenance at every turn.The cellanalyzer calibration object is the standard substance of authoritative department approval.The calibration object of a certain model can only be used to calibrate the instrument of same model, can not be used for the calibration of other brand instruments.The testing result of whole blood cell analysis has only and traces to the source directly or indirectly to reference method, could guarantee result's the accuracy and the comparability of different experiments chamber testing result.The complete blood cell calibration object is the metered dose of haemocyte (liquid) analyser traceability indispensability; As the metrology traceability of cellanalyzer measurement result, estimate and guarantee the clinical trial unique foundation of accuracy as a result.At present, two kinds of methods are adopted in the cellanalyzer calibration usually: 1. 2. new blood stablizes blood or substitute as calibration object.Domestic and international whole blood calibration object great majority employing animal (as bird) haemocyte or macromolecular material (as plastic pellet or nano-substance) product as an alternative are widely used in clinical examination.The complete blood cell calibration object of Shi Yonging just clinically, putting 2～8 ℃, to preserve the terms of validity be not wait in 1～3 month, the uncork rear stability is 1～3 day.
Summary of the invention
The present invention utilizes the combined effect of various kinds of cell active protection liquid and stabilizing agent; Comprise Rb liquid, Wb
10And Wb
22Liquid; Rb liquid is complete blood cell active protection liquid (agent), Wb
10And Wb
22Liquid is combined as the complete blood cell stabilizing agent in appropriate ratio.
1. prescription is formed
(1) complete blood cell active protection liquid (Rb liquid) is made by following component and concentration range: chemical reagent is pure for analyzing; sodium chloride 4.0～6.0g/L, citric acid 0.15～0.3g/L, sodium citrate 1.0～2.0g/L, sodium dihydrogen phosphate 0.5～1.5g/L, adenine 0.1～0.6g/L, glucose 4.0～10.0g/L, sweet mellow wine 4.2～15.0g/L, trehalose 5.0～10g/L, D-mannose 0.5～1.0g/L, glycerine 5.0～10g/L, 1.2-propylene glycol 0.2～1.0g/L, EDTA0.1～0.5g/L, biological bacteriostatic agent an amount of (less than 0.5%).
(2) complete blood cell stabilizing agent I (Wb
10Liquid) make by following component and concentration range: chemical reagent is pure for analyzing, and its concentration range is respectively: Rb liquid 100.0mL, dimethyl sulfoxide 0.1～1.0mL, formaldehyde 1.0～3.0mL, glutaraldehyde 0.01～0.08mL, sodium azide 0.1～0.25g.
(3) complete blood cell stabilizing agent II (Wb
22Liquid) make by following component and concentration range: chemical reagent is pure for analyzing, and its concentration range is respectively: Rb liquid 100.0mL, formaldehyde 1.0～3.0mL, paraformaldehyde 0.05～0.60g, acetaldehyde 0.5～5.0mL, sodium azide 0.1～0.25g.
(4) A liquid, Wb
10And Wb
22After the liquid preparation, (φ 0.2 μ) handles after filtration, puts 2～8 ℃ of refrigerators and preserves use.
2. blood cell analyzer calibrator preparation technology
(1) blood source: healthy people's blood donation of Blood Center or larger animal source property anticoagulated whole blood.Specification is 200mL/ bag or 400mL/ bag blood constituent.
(2) quality requirements: RBC, MCV, HGB, PLT and WBC and classification value thereof are near reference range; Infectiousness indexs such as biological borne causal agent such as hepatitis type B virus (HBsAg), anti-HCV, anti-HIV are all negative.
(3) complete blood cell is separated: get the fresh anticoagulated whole blood of the mankind (or animal derived) (" O " or " A ", " B ", AB blood group) 225mL/ bag, putting horizontal centrifuge 3000 leaves the heart 25～30min and makes haemocyte composition and separating plasma, slowly draw blood plasma, count its volume X mL, the residue blood volume is that (225mL-X) is stand-by.
(4) complete blood cell protection: in about 200mL whole blood, slowly add Wb earlier
10Liquid 18～20mL shakes up gently and puts 2～8 ℃ of 24h and preserve stand-by.
(5) complete blood cell is stable: the complete blood cell through protection is handled slowly adds Wb again
22Liquid 1.0～2.5mL, the limit edged shakes up gently, and fully 2～8 ℃ of preservations are put in sealing behind the mixing, and 4～8h puts upside down gently and makes its abundant mixing for 2～4 times at interval, treats that the reserved sample observing result uses by required ormal weight (0.5mL～5.0mL/ props up) packing after feasible.
(6) points for attention: the essential clean and autoclave sterilization sterilization of employed experiment utensil; The whole blood calibration object prepares overall process must sterile working or carry out in 100,000 grades of environment purifications.
3. quality monitoring:
(1) No. 1 pipe of contrast: get before the blood separation gently fully mixing complete blood cell 2.0mL * 1;
(2) 2～No. 3 pipes of experiment: draw the mixing blood 2.0mL of complete blood cell stabilized treatment * 2 as reserved sample observing;
(3) 1～No. 3 blood samples are respectively on the good cellanalyzer (as serial instruments such as Sysmex, Cell-Dyn, Coulter, MEK, COBAS/ABX, TEK, BC, AC, CA) of calibration, by 1,3,5,7,8～10 results of interval date METHOD FOR CONTINUOUS DETERMINATION of 9....Analyze and the observation of cell change procedure, treat the complete blood cell form stable after, the employing vacuum test tube carries out the quantitative packing of piercing, makes the heparin tube after the packing still keep vacuum state.The whole blood calibration object is put 2～8 ℃, and to preserve time limit be 6 months, and uncork uses 2～8 ℃ of preservations can stablize a week.This product is through biological traceability chain program, the reference method assignment that ICSH or NCCLS provide; The complete blood cell calibration object can be used for the cellanalyzer calibration before the deadline.
The present invention adopts to have histocyte protection and the slowly fixing chemical substance of protein, enzyme and lipid, fresh anticoagulated whole blood cell is exsomatized after, haemocyte still keeps certain metabolism and physiologically active in a long time.Centrifugal blood separates acquisition haemocyte composition, under the synergy of the cytoprotection liquid of proper proportion (as 4: 1 to 5: 1) and cytotostatic agent, activity coordination, complementation and superposition between biological reagent and effect on the one hand, protection liquid provides to be kept the needed nutritional labeling of orthocytosis physiologically active and living environment and is beneficial to and carries out anerobic glycolysis; On the other hand, the cell membrane and the tenuigenin fixed substance that contain low concentration in the stabilizing agent, utilize the combined effect of various kinds of cell active protection liquid and stabilizing agent, the permeation cell membrane interaction is in tenuigenin lentamente, thereby with biomolecule strong bonded such as cell component-protein, enzyme, lipids leucocyte, red blood cell, blood platelet is fixed to some extent effectively and effectively protection; Make haemocyte keep its mode of appearance feature.Therefore, the haemocyte of handling well is through biological traceability chain program and reference method assignment thereof; Whole blood quality control materials or calibration object before the deadline, can be used as the calibration object of cellanalyzer, when adding supporting hemolytic agent of instrument and dilution, red blood cell in the calibration object can dissolve rapidly, and the effect that classification was counted and reached to cellanalyzer is satisfied in apparent volume and inner structure generation significant change under the effect of leucocyte surfactant in being subjected to hemolytic agent.
The complete blood cell calibration object viscosity of the present invention's development is low, and there is not matrix effect in mixing easily; The vacuum seal postposition is preserved time limit for 2～8 ℃ and was at least 6 months, and uncork uses 2～8 ℃ of preservations can stablize a week, and it is 36h that room temperature (15 ℃～25 ℃) can be preserved the term of validity.
The present invention adopts human or animal derived complete blood cell; utilize the synergistic combination effect of various kinds of cell active protection liquid and stabilizing agent; the complete blood cell calibration object of handling well through cytoprotection and stabilization technique has good stable; not only can cell dyeing and microscopic counting, and the more clinical existing whole blood calibration object of the term of validity (thing) or Quality Control thing (product) are long.
Embodiment
Cytoactive protection liquid of the present invention and stabilizing agent comprise protection liquid (agent), Wb
10And Wb
22Liquid.
Prescription is formed
(1) complete blood cell active protection liquid (Rb liquid): sodium chloride 5.0g/L, citric acid 0.2g/L, sodium citrate 2.0g/L, sodium dihydrogen phosphate 0.75g/L, adenine 0.3g/L, glucose 7.5g/L, sweet mellow wine 10.0g/L, trehalose 7.8g/L, D-mannose 0.6g/L, glycerine 8.0g/L, 1.2-propylene glycol 0.4g/L, EDTA0.25g/L, biological bacteriostatic agent is an amount of.2～8 ℃ of refrigerators are preserved standby.
(2) complete blood cell stabilizing agent I (Wb
10Liquid): Rb liquid 100.0mL, dimethyl sulfoxide 0.5mL, formaldehyde 3.0mL, glutaraldehyde 0.05mL, sodium azide 0.15g.2～8 ℃ of refrigerators are preserved standby.
(3) complete blood cell stabilizing agent II (Wb
22Liquid): Rb liquid 100.0mL, formaldehyde 1.0mL, paraformaldehyde 0.25g, acetaldehyde 4.5mL, sodium azide 0.2g.2～8 ℃ of refrigerators are preserved standby.
Cellanalyzer of the present invention is as follows with the calibration object production technology:
(1) complete blood cell is separated: get the fresh anticoagulated whole blood of the mankind (or animal derived) (" O " or " A ", " B ", AB blood group) 225mL/ bag, putting horizontal centrifuge 3000 leaves the heart 25～30min and makes haemocyte composition and separating plasma, slowly draw blood plasma, count its volume X mL (X is for being 20-28mL), the residue blood volume is that (225mL-X) is stand-by.Sampling 2.0mL is No. 1 control tube behind the mixing.
(2) complete blood cell protection: in above-mentioned 225mL-X whole blood, slowly add Wb earlier
10Liquid 18～20mL shakes up gently and puts 2～8 ℃ of 24h and preserve stand-by.Sampling 2.0mL is No. 2 pipes behind the mixing.
(3) complete blood cell is stable: the complete blood cell through protection is handled slowly adds Wb again
22Liquid 1.0～2.5mL, the limit edged shakes up gently, and fully 2～8 ℃ of preservations are put in sealing behind the mixing, and 4～8h puts upside down gently and makes its abundant mixing for 2～4 times at interval, treats that the feasible back of reserved sample observing result is by required ormal weight packing use.Sampling 2.0mL is No. 3 pipes behind the mixing.
(4) 1～No. 3 pipe of quality monitoring is on the good Sysmex kx-21 cellanalyzer of calibration, by 1,3,5,7,8～10 results of interval date METHOD FOR CONTINUOUS DETERMINATION of 9....5 index results of blood analysis such as histogram and WBC, RBC, MCV/HCT, HGB, PLT and histogram quality inspection requirement qualified or up to specification can vacuum be distributed into product.
(5) vacuum packing: make a cover piercing quantitative packing frock according to actual needs, behind the abundant mixing of whole blood calibration object, adopt vacuum test tube (2.0～5.0mL specification) to carry out the quantitative packing of piercing, make the heparin tube after the packing still keep vacuum state.
(6) traceability assignment: by a continual relatively chain with regulation uncertainty, the characteristic that the value of measurement result or measurement standard can be connected with the normative reference or the method for regulation.Normally set up a cover reference measure program, the reference method that provides according to ICSH or NCCLS carries out assignment to the whole blood calibration object.
(7) preserve and clinical use: 2～8 ℃ of preservations of whole blood calibration object vacuum seal postposition, use for clinical cellanalyzer calibration, have time limit to be at least 6 months, uncork uses 2～8 ℃ of preservations can stablize a week, and it is 36h that room temperature (15 ℃～25 ℃) can be preserved the term of validity.
Claims (2)
1, a kind of blood cell analyzer calibrator is characterized in that: utilize the synergistic combination effect of various kinds of cell active protection liquid and stabilizing agent, comprise Rb liquid, Wb
10And Wb
22Liquid; Rb liquid is complete blood cell active protection liquid, Wb
10And Wb
22Liquid is combined as the complete blood cell active stabilizer in proportion;
(1), Rb liquid makes by following component and concentration range: sodium chloride 4.0～6.0g/L, citric acid 0.15～0.3g/L, sodium citrate 1.0～2.0g/L, sodium dihydrogen phosphate 0.5～1.5g/L, adenine 0.1～0.6g/L, glucose 4.0～10.0g/L, sweet mellow wine 4.2～15.0g/L, trehalose 5.0～10g/L, D-mannose 0.5～1.0g/L, glycerine 5.0～10g/L, 1.2-propylene glycol 0.2～1.0g/L, EDTA0.1～0.5g/L, biological bacteriostatic agent is an amount of;
(2), the complete blood cell stabilizing agent is made by following component and concentration range:
Wb
10Liquid: Rb liquid 100.0mL, dimethyl sulfoxide 0.1～1.0mL, formaldehyde 1.0～3.0mL, glutaraldehyde 0.01～0.08mL, sodium azide 0.1～0.25g;
Wb
22Liquid: Rb liquid 100.0mL, formaldehyde 1.0～3.0mL, paraformaldehyde 0.05～0.60g, acetaldehyde 0.5～5.0mL, sodium azide 0.1～0.25g.
2, a kind of preparation technology of blood cell analyzer calibrator as claimed in claim 1 is characterized in that, is made by following production technology:
(1) complete blood cell is separated: get the fresh anticoagulated whole blood cell of the mankind (or animal derived) (" O " or " A ", " B ", AB blood group) 225ml/ bag, after putting horizontal centrifuge 3000 and leaving the heart 25～30min and make haemocyte composition and separating plasma, slowly draw small amount, count its volume X ml, the residue blood volume is that 225ml-X is stand-by;
(2), complete blood cell protection: in above-mentioned 225ml-X whole blood, slowly add Wb earlier
10Liquid 18～20mL shakes up gently and puts 2～8 ℃ of 24h and preserve stand-by;
(3) complete blood cell is stable: the complete blood cell through protection is handled slowly adds Wb again
22Liquid 1.0～2.5mL, the limit edged shakes up gently, and fully 2～8 ℃ of preservations are put in sealing behind the mixing, and 4～8h puts upside down gently and makes its abundant mixing for 2～4 times at interval, and the final vacuum packing is used;
(4) blood cell analyzer calibrator is imitated phase stability: the whole blood calibration object after the vacuum seal is put 2～8 ℃ and is preserved time limit and be at least 6 months, and uncork uses rearmounted 2～8 ℃ of preservations can stablize a week, and it is 24h that room temperature (15 ℃～25 ℃) can be preserved the term of validity.
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| CN200910186118.7A CN101672853B (en) | 2009-09-28 | 2009-09-28 | Blood cell analyzer calibrator and preparation process thereof |
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| US4179398A (en) * | 1977-03-21 | 1979-12-18 | ICN Medical Laboratories, Inc. | Platelet control composition |
| US4698312A (en) * | 1986-07-28 | 1987-10-06 | Fisher Scientific Company | Stabilizing blood cells with aromatic polyaldehyde for use in hematology controls and calibrators |
| US20030013074A1 (en) * | 2000-12-04 | 2003-01-16 | Kenzo Bamba | Cell-preservation liquid and method of preserving cells by using the liquid |
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