CN101852694A - Sputum smear used for acid-fast bacillus microscope examination in proficiency testing activity and preparation method and application thereof - Google Patents
Sputum smear used for acid-fast bacillus microscope examination in proficiency testing activity and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a sputum smear used for an acid-fast bacillus microscope examination in a proficiency testing activity, which comprises a negative acid-fast bacillus sputum smear, a 1<+> acid-fast bacillus sputum smear, a 2<+> acid-fast bacillus sputum smear, a 3<+> acid-fast bacillus sputum smear and a 4<+> acid-fast bacillus sputum smear, wherein the acid-fast bacillus content of the negative sputum smear is 0 per 300 visual fields; the acid-fast bacillus content of the 1<+> sputum smear is 3 to 9 per 100 visual fields; the acid-fast bacillus content of the 2<+> sputum smear is 1 to 9 per 10 visual fields; the acid-fast bacillus content of the 3<+> sputum smear is 1 to 9 per one visual field; the acid-fast bacillus content of the 4<+> sputum smear is more than 9 per one visual field; and the anti-fast bacilli are derived from sputamentum or a culture bacterial colony. Besides, the invention also discloses a preparation method and application of the sputum smear. The sputum smear is proved to have high uniformity and stability, and can be applied to a tuberculosis detection proficiency testing activity or laboratory internal and external quality controls.
Description
Technical field
The present invention relates to health quarantine, health supervision, medical technology, public health field, specifically relate to a kind of phlegm smear, relate in particular to a kind of phlegm smear that is used for the microexamination of ability verification acid-fast bacilli; In addition, the invention still further relates to the preparation method and the purposes of this phlegm smear.
Background technology
As the member state of WTO, in April, 2002, China set up the conformity assessment system of becoming compatible with internationally accepted practices, comprising laboratory authorized by state system.
The basic role of setting up quality management system is the data and the report that help the laboratory to provide to continue to meet the demands, enhances competitiveness, strengthens Customer Satisfaction.(1) laboratory needs quality management system for the service that client continues to provide satisfied.The quality management system method is encouraged the lab analysis customer requirement, and regulation satisfies the detection/calibration implementation procedure and the relevant supporting process of customer requirement, and it is controlled that it is continued.Quality management system can provide the framework of Continual Improvement, to increase client and the satisfied chance of other related side.(2) quality management system also is client's needs.Client can select satisfied supplier by the ability of quality management system evaluation experimental chamber.
One, proficiency testing
Proficiency testing (Proficiency Testing) is to utilize between the laboratory comparison to judge the activity of laboratory and inspection body ability, also is that accreditation body adds and keeps the world one of the necessary condition of agreement (MRA) of recognizing each other.
Quality evaluation is the process that basis and other network laboratories compare batch testing and blind method reinspection evaluation of result laboratory ability and operational quality between the chamber.
Indoor quality control is meant the internal check and the monitoring of each operating process of inside, laboratory.
Authentication is meant the conformity assessment activity that is met the Compulsory Feature or the standard of correlation technique standard, correlation technique standard by certification authority's proof product, service, management system.
Approval is by authoritative institution the program of being duly admitted to be made in detection/calibration that detection/calibration laboratory and personnel thereof have the ability to carry out particular type.So-called authoritative institution is meant the responsibility with law or administrative authorization and the government or the non-government institution of power.Thisly admit, mean to admit to detect/calibration laboratory has managerial ability and technical capability to be engaged in the work of specific area.Thereby the essence of Laboratory Accreditation is the approval of specific detection/calibration item that the laboratory is carried out, is not breadboard all business activities.The chamber approval that experimentizes can improve the management level and the technical capability in laboratory self, guarantees to provide the accuracy and the reliability of data, increases client to breadboard trust.Particularly, can reduce the following aspects:
(1) shows that the laboratory has possessed the technical capability of carrying out calibration/detection by relevant international rule.
(2) strengthen the competitiveness of laboratory, win the trust of government department and various circles of society in calibration/detection market.
(3) participate in international Laboratory Accreditation bilateral and multi-lateral cooperation, admitted widely.
(4) list " National Laboratory's approval register " in, improve breadboard popularity.
(5) can in the approval scope of project, use approval mark.
Requirement according to CNAL (CNAL), the condition that the detection/calibration laboratory of application approval must satisfy comprises: have clear and definite legal status, promptly laboratory or place parent should be the entities that can independently bear legal responsibility; Set up quality management system by accreditation criteria and application note thereof, and each key element (process) moved all and respective record has been arranged, comprised complete internal check and management review; Quality management system operation at least six months; Can accept the on-site review of CNAL in back three months in application; Have the detection/rated capacity in the application approval scope, and may the time participated in CNAL or its ability verification of admitting at least; Power with domination resource requirement; Observe relevant regulations such as CNAL approval rule, approval policy, comprise the payment approved charge, fulfil relevant obligation.
CNAL can provide comprehensive approval to the laboratory, comprises the laboratory that product or material are monitored, tested or estimate, and the laboratory that detecting instrument or measurement mechanism are calibrated.Laboratory Accreditation mechanism makes a promise there is not any discriminatory behavior in application accredited laboratory, no matter i.e. its attribute, for privately owned, share-holding system, industry or government, no matter also what of its personnel amount, the size of scale or the size of detection/calibration activities scope all provide accredited services alike.
The four principles of Laboratory Accreditation employing is voluntary application, non-discrimination, experts' evaluation and authorized by state in the world.
The application principle is meant voluntarily: whether the laboratory applies for approval, and according to the autonomous decision of its demand, promptly accreditation body can not force any one laboratory application.But for the laboratory, in fact this voluntary principle can be subjected to the restriction of customer demand, promptly must can to bear detections/calibration by approval professional when client proposes the laboratory, and this is wished to accept when professional in the laboratory, applies for approving just becoming a kind of coercive action; In addition, when breadboard parent mechanism or its management organization when requiring, the application approval also can become a kind of mandatory requirement.No matter based on above-mentioned any situation, compulsory requirement can be from accreditation body.
For detection/calibration laboratory, should select ISO/IEC 17025 Laboratory Accreditation.
Two, the acid-fast bacilli detection is searched in the acid-fast stain of phlegm smear
" acid-fast stain of phlegm smear is searched acid-fast bacilli and detected " is that efficient in the tuberculosis bacteriology checking-cost is than the highest detection technique as the important method of finding the infection sources lungy, determine diagnosis and formulate chemotherapy regimen, examination curative effect, estimate prevention effect.The tuberculosis bacteriological detection is the important component part of tuberculosis controlled target, for strengthening the tuberculosis Control work, realizes that global tuberculosis controlled target is significant, plays indispensable vital role in modern tuberculosis Control work." acid-fast stain of phlegm smear is searched acid-fast bacilli and detected " also is the most important project in the tuberculosis laboratory examination, carrying out the acid-fast stain of phlegm smear, to search the ability verification of acid-fast bacilli test item be to provide foundation for Laboratory Accreditation that this project is carried out in the laboratory with this test item, also is to help the laboratory that data and the report that continues to satisfy tuberculosis laboratory diagnosis requirement, a kind of important channel of enhancing competitiveness, strengthening Customer Satisfaction are provided.Implement this project ability verification and not only can understand the level of laboratory on this detectability that participate in evaluation and electing, promote the acid-fast stain operative technique, the standardization of microscope inspection survey technology, to continue to improve breadboard work efficiency and reliability, and improve and improve constantly laboratory phlegm smear acid-fast stain by adopting a series of measures and search the ability that acid-fast bacilli detects, reinforcement is to the supervision and management of tuberculosis test experience chamber, improve the level of China tuberculosis test experience chamber, guarantee that the quality that acid-fast bacilli detects is searched in the acid-fast stain of phlegm smear in the tuberculosis bacteriological detection, promote the development of tuberculosis prevention and treatment work.The intercommunion platform of a technology, quality management also is provided in the work of phlegm smear detection acid-fast bacilli project for laboratory, domestic each department.
Acid-fast bacilli (acid-fast bacillus, AFB) mainly refer to mycobacterium because the singularity of eucaryotic cell structure and cell component, have the characteristic that can tolerate the acid medium decolouring after basic dyeing, the bacterium that possesses corresponding dyeing characteristic is called as acid fast bacteria traditionally.
Ziehi-Neelsen stain is meant that acid fast bacteria has the biological character of tolerance acid medium decolouring, this bacterioid is under the synergy of carbolic acid (phenol), painted by the azaleine coloring agent, can tolerate the acidic ethanol decolouring, keep aubergine during microscopic examination, and the non-acid fast bacteria in other cast-off cells or the sample is by after the acidic ethanol decolouring, can be dyed by the counterstain methylenum careuleum to be blueness.In honor of inventor's Ziehi-Neelsen stain of method also claims the Ziehl-Neelson decoration method, and the Z-N decoration method is luxuriant-Nissl's staining.Acid-fast bacilli comprises that mainly mycobacterium and minority have the bacterium of acid-fast stain feature.
Mycobacterium comprises mycobacterium tuberculosis complex and non-tuberculous mycobacteria.The Much's bacillus majority is shaft-like, crooked slightly; Thalline width 0.3-0.6 μ m (micron); Thalline length differs, and is between 0.5-8 μ m, most at 1.5-3.5 μ m; The minority thalline is than elder's shape in the shape of a spiral; Dye in the good Much's bacillus thalline, can find painted darker Babes-Ernst bodies.Contain the more fresh specimens of Much's bacillus through the microscopically of acid-fast stain, can find the bacterium of single existence, also can see the bacterium that assembles cluster or branch-like arrangement 1000 times of amplifications.The form of most non-tuberculous mycobacterias is short and thick than Much's bacillus, is bar-shaped, corynebacterium, even graininess.
The phlegm smear is meant with human body oozy sputum directly or after treatment, gets on a certain amount of microslide that evenly is applied in cleaning the phlegm film that forms after the drying.Qualified sputum is that human body is after deep breathing, by the secretion of deep lung expectoration.
The purpose of acid-fast bacilli microexamination: 1, the diagnosis index of infectiousness pulmonary tuberculosis, 2, estimate infectiousness chemotherapy effect lungy, 3, serve for the tuberculosis epidemiology index.
The bottleneck that the key that the ability verification of acid-fast bacilli test item carries out is searched in restriction phlegm smear acid-fast stain at present is exactly the preparation of sample, and its difficulty is:
1, the mycobacterium bacterial suspension is the suspension of particle, and it is difficult point that the homogeneity that how to guarantee each sample in extensive specimen preparation process meets the requirements.
2, to search that acid-fast bacilli detects be a semiquantitative microbial morphology project to the acid-fast stain of phlegm smear, its diagnosis basis is to give semiquantitative testing result according to observing the quantity that contains acid-fast bacilli in the average visual field in the phlegm smear after 1000 times of amplifications of microscopically, does not find acid-fast bacilli-report as 0/300 visuals field: feminine gender; Find 1-2 bar acid-fast bacilli/300 visual field-reports: suspicious; Find 3-9 bar acid-fast bacilli/100 visual field-report: 1+; Find 1-9 bar acid-fast bacilli/10 visual field-report: 2+; Find the 1-9 bar acid-fast bacilli/visual field-report a: 3+; Find＞9 acid-fast bacillis/visual field-report a: 4+; The consistent of sxemiquantitative concentration that how to guarantee each sample in the extensive sample is difficult point.The acid-fast stain of phlegm smear is searched acid-fast bacilli examining report mode and is checked (refilling member) referring to [GB15987-1995] infectiousness pulmonary tuberculosis diagnostic criteria and treatment principle appendix B phlegm tubercle bacillus.
3, because test result of samples derives from the bacteria content in the average visual field, thus subjective factor outwardness, how to overcome the influence of subjective factor to proficiency testing result's assessment, be difficult point.
Because the difficulty that specimen preparation and result judge, quality evaluation is searched between checking activity of acid-fast bacilli detectability or chamber in the phlegm smear acid-fast stain of various sxemiquantitative concentration or indoor quality control was not carried out in China so cover.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of phlegm smear that is used for the microexamination of ability verification acid-fast bacilli.The homogeneity of this phlegm smear and stability are higher, adopt this phlegm smear can be applied in the work of quality evaluation between checking activity of tuberculosis detectability or chamber, laboratory, indoor quality control, thereby acid-fast stain is searched acid-fast bacilli and is detected the training that this project is carried out quality control and technician to the phlegm smear.For this reason, the present invention also provides the preparation method and the purposes of this phlegm smear.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of phlegm smear that is used for the microexamination of ability verification acid-fast bacilli is provided, it comprises feminine gender, 1+, 2+, 3+, the 4+ phlegm smear of totally five kinds of acid-fast bacilli content, and the acid-fast bacilli content of this feminine gender phlegm smear is 0/300 visuals field; The acid-fast bacilli content of this 1+ phlegm smear is 3-9 bar acid-fast bacilli/100 visuals field; The acid-fast bacilli content of this 2+ phlegm smear is 1-9 bar acid-fast bacilli/10 visuals field; The acid-fast bacilli content of this 3+ phlegm smear is a 1-9 bar acid-fast bacilli/visual field; The acid-fast bacilli content of this 4+ phlegm smear is＞9 an acid-fast bacillis/visual field; Described acid-fast bacilli derives from sputum or culture of bacteria bacterium colony.
In another aspect of this invention, provide a kind of preparation method of above-mentioned phlegm smear, the acid-fast bacilli that described phlegm smear contains derives from sputum, and this method comprises the steps:
1) get the phlegm in morning of different tuberculosis patients, be positioned in the aseptic sputum specimen collection container, bacteria inactivation is handled (can adopt 80 ℃ of water-baths, 30 minutes-120 minutes);
2) behind the natural cooling, contain in aseptic sputum is handled pipe, make sputum liquefaction and with after the PBS dilution, centrifugal, make bacterium concentrate on the test tube bottom, abandon supernatant, add the PBS mixing in the sediment as the phlegm suspension, as initial grade concentration, calculate extension rate according to acid-fast bacilli purpose grade concentration with phlegm smear acid-fast stain sediments microscope inspection method bacterial detection quantity then, and be diluted to the phlegm dilution of purpose grade concentration with PBS;
3) get above-mentioned phlegm dilution and on microslide, smear ovalization phlegm film, air dry, flame is fixed; Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, room temperature preservation.
In step 2) in, the purpose grade concentration of acid-fast bacilli is 0/ml in the described phlegm dilution, 0.8-2.2 * 10
4Bar/ml, 0.3-2.2 * 10
5Bar/ml, 0.3-2.2 * 10
6Bar/ml, perhaps＞2.2 * 10
6Bar/ml.
After step 3) is finished, also comprise following packaging step: the phlegm smear adopts three layers of packing, and ground floor is placed on the phlegm smear in the special-purpose Glass carrier box, and the second layer is at Glass carrier box external application antidetonation barrier film parcel, and the 3rd layer is external application waterproof paper case packing.
In the step 3), described sample number into spectrum is that computer programming designs one group of random sequence number that does not have the repetition numeral, should numeral corresponding to each phlegm smear, make each sample obtain unique Digital ID, numeral preparation area, preparation time and phlegm smear sequence number in this Digital ID.
In another aspect of this invention, provide the preparation method of another kind of above-mentioned phlegm smear, the acid-fast bacilli that described phlegm smear contains derives from the culture of bacteria bacterium colony, and this method comprises the steps:
1) preparation of bcg bacteria suspension: the bacterial clump of getting two weeks of first growth of Bacille Calmette-Guerin is put into glass bacteria grinder bottom or is had the centrifuge tube of beaded glass, wherein add the PBS that contains 0.5% Tween-80, handle (can adopt 80 ℃ of water-baths, 30 minutes-120 minutes) through bacteria inactivation; Behind the natural cooling, fully stir vibration and make bacterium liquid be the cheese sample, with physiological saline or PBS mill bacterium and the dilution that contains 0.5% Tween-80, with standard Maxwell opacity tube than turbid, be made into the bacteria suspension of 1mg/ml;
2) get the phlegm in morning of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (can adopt 80 ℃ of water-baths, 30 minutes-120 minutes) through bacteria inactivation;
3) behind the natural cooling, contain in aseptic sputum is handled pipe, make sputum liquefaction and with the PBS dilution, centrifugal, make that solids concentrates on the test tube bottom in the phlegm, abandon supernatant, the PBS mixing of sputum such as addings volume is as the phlegm suspension in the sediment;
4) use phlegm smear acid-fast stain sediments microscope inspection method bacterial detection quantity as initial grade concentration, calculate extension rate according to acid-fast bacilli purpose grade concentration then, what the bacteria suspension that the phlegm suspension made from step 3) is made step 1) was diluted to purpose grade concentration contains bacterium phlegm dilution; Get the above-mentioned bacterium phlegm dilution that contains and on microslide, smear ovalization phlegm film, air dry, flame is fixed; Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, room temperature preservation.
In step 4), the described purpose grade concentration that contains acid-fast bacilli in the bacterium phlegm dilution is 0/ml, 0.8-2.2 * 10
4Bar/ml, 0.3-2.2 * 10
5Bar/ml, 0.3-2.2 * 10
6Bar/ml, perhaps＞2.2 * 10
6Bar/ml.
After step 4) is finished, also comprise following packaging step: the phlegm smear adopts three layers of packing, and ground floor is placed on the phlegm smear in the special-purpose Glass carrier box, and the second layer is at Glass carrier box external application antidetonation barrier film parcel, and the 3rd layer is external application waterproof paper case packing.
In the step 4), described sample number into spectrum is that computer programming designs one group of random sequence number that does not have the repetition numeral, should numeral corresponding to each phlegm smear, make each sample obtain unique Digital ID, numeral preparation area, preparation time and phlegm smear sequence number in this Digital ID.
In another aspect of this invention, provide a kind of uniformity detecting method of above-mentioned phlegm smear, comprise the steps:
1) from the phlegm smear of same levels concentration, randomly draws and need the phlegm of quantity smear, with Ziehi-Neelsen stain the phlegm smear of sampling is carried out acid-fast stain, and each the phlegm smear that extracts carried out microscopic examination, the quantity of observed acid-fast bacilli under numeration and 1-300 visual field of report;
2) testing result is for judging the rank value that obtains according to the acid-fast bacilli amount in each phlegm smear: feminine gender, 1+, 2+, 3+, 4+; Testing result and expected results are compared; Negative phlegm smear: sampling phlegm smear results is all negative, illustrates that then population sampled is even; 1+ phlegm smear: sampling phlegm smear results is 1+ or 2+ or 1-8 bar/300 visuals field, illustrates that then population sampled is even; 2+ phlegm smear: sampling phlegm smear results is 1+ or 2+ or 3+, illustrates that then population sampled is even; 3+ phlegm smear: sampling phlegm smear results is 2+ or 3+ or 4+, illustrates that then population sampled is even; 4+ phlegm smear: sampling phlegm smear results is 3+ or 4+, illustrates that then population sampled is even; Negative findings appears in " 1+ " above phlegm smear, illustrates that population sampled is inhomogeneous.
In another aspect of this invention, provide a kind of Detection of Stability method of above-mentioned phlegm smear, comprise the steps:
1) randomly draw and need the phlegm of quantity smear, be delivered to the laboratory, different regions after the packing soon, room temperature preservation is returned in the express delivery mode to planning Return Date, to after returning the phlegm smear and carrying out acid-fast stain, searches acid-fast bacilli with microscopic method with Ziehi-Neelsen stain; Perhaps randomly drawing needs the phlegm of quantity smear, carries out being delivered to the laboratory, different regions soon after the acid-fast stain of phlegm smear with Ziehi-Neelsen stain, and room temperature preservation is returned in the express delivery mode to planning Return Date, searches acid-fast bacilli with microscopic method;
2) result that will detect and expected results compare, and negative phlegm smear: testing result is all negative, and phlegm smear qualified stability then is described; 1+ phlegm smear: testing result is 1+ or 2+ or 1-8 bar/300 visuals field, and phlegm smear qualified stability then is described; 2+ phlegm smear: testing result is 1+ or 2+ or 3+, and phlegm smear qualified stability is described; 3+ phlegm smear: testing result is 2+ or 3+ or 4+, and phlegm smear qualified stability is described; 4+ phlegm smear: testing result is 3+ or 4+, and phlegm smear qualified stability is described; Negative findings appears in " 1+ " above phlegm smear, and phlegm smear instability is described; If the result is variant, phlegm smear instability is described, need search the unsettled reason of phlegm smear, prepare the phlegm smear again or change storage, means of transportation.
In another aspect of this invention, provide the application of a kind of above-mentioned phlegm smear in proficiency testing.
In another aspect of this invention, provide the application in quality evaluation and the indoor quality control between the chamber, laboratory of a kind of above-mentioned phlegm smear.
The present invention is used for the phlegm smear of ability verification acid-fast bacilli microexamination, is the key of carrying out the ability verification of " acid-fast stain of phlegm smear is searched acid-fast bacilli and detected " project.The phlegm smear that is used for the microexamination of ability verification acid-fast bacilli is with the difference of other type phlegm smear: 1, it must be made up of the phlegm smear of different acid-fast bacilli content, 2, the phlegm smear quantity of each acid-fast bacilli content wants the acid-fast bacilli quantity between of certain scale and each phlegm smear to want evenly, can satisfy the requirement of ability verification, 3, the stability of phlegm smear wants to satisfy the requirement of ability verification, and 4, the bio-safety aspect of phlegm smear wants to satisfy the requirement of ability verification.The preparation of carrying out proficiency testing phlegm smear can guarantee that this project proficiency testing is implemented.
Beneficial effect of the present invention is: in year February in April, 2009-2010, the applicant has adopted method for preparing phlegm smear has carried out the ability verification of " acid-fast stain of phlegm smear is searched acid-fast bacilli and detected " to the whole nation 43 tame specialized laboratories.The result shows that this proficiency testing sends 440 test sample altogether, has repaid 430 test result of samples, has 414 sample detection results correct, accounts for 96.3% of repayment sample number.In repayment result's 43 tame laboratories, there are 30 tame laboratories to obtain and get a mark of 100,10 tame laboratories obtain 95 fens, and 1 tame laboratory obtains 90 fens, and 2 tame laboratories obtain 85 fens.The chamber of the participating in the experiment scoring of 43 families is all more than 80 minutes, and is all qualified.Have the situation that do not meet that low false positive (4) and quantization error (12) appear in 16 testing results, relate to 13 tame laboratories.Do not meet and exist the higher situation of systematicness to relate to 3 tame laboratories among the result.Wherein there is 1 low false positive erroneous judgement in 4 negative sample among the 9038 laboratories repayment result, has 2 quantization error erroneous judgements in 6 positive sample.The presentation of results of this proficiency testing the preparation method of phlegm smear of the present invention be science, reasonably also be enforceable that and homogeneity and stability meets the requirement of ability verification, is that a kind of practicality very strong phlegm smear and phlegm are coated with piece preparation method.
Description of drawings
Fig. 1 is that 4+ phlegm smear amplifies 1000 times synoptic diagram at microscopically among the embodiment after acid-fast bacilli dyeing;
Fig. 2 is that 3+ phlegm smear amplifies 1000 times synoptic diagram at microscopically among the embodiment after acid-fast bacilli dyeing;
Fig. 3 is that 2+ phlegm smear amplifies 1000 times synoptic diagram at microscopically among the embodiment after acid-fast bacilli dyeing;
Fig. 4 is that 1+ phlegm smear amplifies 1000 times synoptic diagram at microscopically among the embodiment after acid-fast bacilli dyeing;
Fig. 5 is that negative phlegm smear amplifies 1000 times synoptic diagram at microscopically among the embodiment after acid-fast bacilli dyeing.
Embodiment
Following examples only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, or the condition of advising according to manufacturer.
1. purpose
The purpose of carrying out this laboratory detectability checking work is to understand the integral level of this detection range, and the result of this proficiency testing is that objectively responding of acid-fast bacilli detectability searched in laboratory phlegm smear acid-fast stain.By this proficiency testing project implementation, can strengthen the laboratory is carried out the supervision and management of tuberculosis pathogen detection work, promote the development of tuberculosis prevention and treatment work.
2. experimental program
ISO/IEC guide rule 43-1:1997 " utilizing the proficiency testing that contrasts between the laboratory " and GB/T15483.1-1999 " utilizing the proficiency testing that contrasts between the laboratory " are followed in the design of these proficiency testing plans.The uniqueness coding is carried out in each chamber of participating in the experiment.The laboratory code only appears in report, thus maintaining secrecy for information about to the chamber of participating in the experiment.
2.1 the design of sample
This proficiency testing scheme prepares 10 groups of samples of variable concentrations, 3 groups of wherein negative acid-fast bacilli concentration, 3 groups of 1+ acid-fast bacilli concentration, 2 groups of 2+ acid-fast bacilli concentration, 1 group of 3+ acid-fast bacilli concentration, 1 group of 4+ acid-fast bacilli concentration, 150 samples that bacteria containing amount is identical are arranged in every group, give every sample exclusive number.From 10 groups of samples, respectively randomly draw a test sample, form the test sample group, be distributed to the chamber of participating in the experiment.Respectively the test sample numbering received of the chamber of participating in the experiment is different, guarantees the confidentiality of sample, reflects each breadboard detection level strictly according to the facts, has guaranteed the seriousness of proficiency testing.
2.2 the preparation of sample
2.2.1, the preparation of microslide
New microslide is soaked in 95% the ethanol, spends the night, take out air dry, standby.
2.2.2, the preparation method of negative phlegm smear
Negative sample preparation method: get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes-120 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, NaOH-NALC (NaOH-N acetylcysteine) mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8 PBS (phosphate buffer), centrifugal 3000g (centrifugal force), 30 minutes, make in the phlegm solids concentrate on the test tube bottom, abandoning the supernatant taking precipitate adds and the isopyknic PBS mixing of sputum specimen, get the phlegm of above-mentioned concentration and handle the suspension mixing, after get 30 μ l in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be prepared into negative phlegm smear, be positioned in the sample box room temperature preservation.
2.2.3, the preparation method of positive spit smear
Embodiment 1 acid-fast bacilli derives from the preparation method of sputum
A) preparation method of 1+ phlegm smear
Get the phlegm in morning (disease prevention and control center, Shanghai provides) of different tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes was diluted to 50ml with PH6.8PBS, centrifugal 3000g after 20 minutes, 30 minutes, make tulase concentrate on the test tube bottom, abandon supernatant, add 1ml PBS mixing in the sediment as the phlegm suspension, detect wherein bacterial number with acid-fast stain sediments microscope inspection method, being diluted to final concentration with PBS is 2.2 * 10
4The phlegm dilution of bar/ml is got above-mentioned phlegm dilution 30 μ l in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
B) preparation method of 2+ phlegm smear
Get the phlegm in morning (disease prevention and control center, Shanghai provides) of different tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 120 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes was diluted to 50ml with PH6.8PBS, centrifugal 3000g after 20 minutes, 30 minutes, make tulase concentrate on the test tube bottom, abandon supernatant, add 1ml PBS mixing in the sediment as the phlegm suspension, detect wherein bacterial number with acid-fast stain sediments microscope inspection method, being diluted to final concentration with PBS is 2.2 * 10
5The phlegm dilution of bar/ml is got above-mentioned phlegm dilution 30 μ l in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
C) preparation method of 3+ phlegm smear
Get the phlegm in morning (disease prevention and control center, Shanghai provides) of different tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 80 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes was diluted to 50ml with PH6.8PBS, centrifugal 3000g after 20 minutes, 30 minutes, make tulase concentrate on the test tube bottom, abandon supernatant, add 1ml PBS mixing in the sediment as the phlegm suspension, detect wherein bacterial number with acid-fast stain sediments microscope inspection method, being diluted to final concentration with PBS is 2.2 * 10
6The phlegm dilution of bar/ml is got above-mentioned phlegm dilution 30 μ l in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
D) preparation method of 4+ phlegm smear
Get the phlegm in morning (disease prevention and control center, Shanghai provides) of different tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes was diluted to 50ml with PH6.8 PBS, centrifugal 3000g after 20 minutes, 30 minutes, make tulase concentrate on the test tube bottom, abandon supernatant, add 1ml PBS mixing in the sediment as the phlegm suspension, detect wherein bacterial number with acid-fast stain sediments microscope inspection method, being diluted to final concentration with PBS is 2.3 * 10
6The phlegm dilution of bar/ml is got above-mentioned phlegm dilution 30 μ l in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
Embodiment 2 acid-fast bacillis derive from the preparation method of culture of bacteria bacterium colony
1, the preparation of bcg bacteria suspension
Get the bacterial clump in two weeks of first growth of the Bacille Calmette-Guerin that Shanghai Vaccine and Serum Institute provides and put into glass bacteria grinder bottom, wherein add the PBS that 100 μ l contain 0.5% Tween-80, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, insert mill bacterium rod and twist to make and be the cheese sample, containing the physiological saline mill bacterium dilution of 0.5% Tween-80, with standard Maxwell opacity tube (MacFarland No.1) than turbid, be made into the bacteria suspension of 1mg/ml.
2, the preparation of positive spit smear
A) preparation method of 1+ phlegm smear
Get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 70 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8PBS, centrifugal 3000g 30 minutes, makes in the phlegm solids concentrate on the test tube bottom, abandon supernatant, adding waits the PBS mixing of sputum volume as the phlegm suspension in the sediment; With above-mentioned phlegm suspension the bacteria suspension of 1mg/ml being diluted to final concentration is 0.8 * 10
4Bar/ml contains bacterium phlegm dilution, gets the above-mentioned bacterium phlegm dilution 30 μ l that contain in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
B) preparation method of 2+ phlegm smear
Get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8PBS, centrifugal 3000g 30 minutes, makes in the phlegm solids concentrate on the test tube bottom, abandon supernatant, adding waits the PBS mixing of sputum volume as the phlegm suspension in the sediment; With above-mentioned phlegm suspension the bacteria suspension of 1mg/ml being diluted to final concentration is 0.3 * 10
5Bar/ml contains bacterium phlegm dilution, gets the above-mentioned bacterium phlegm dilution 30 μ l that contain in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
C) preparation method of 3+ phlegm smear
Get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8PBS, centrifugal 3000g 30 minutes, makes in the phlegm solids concentrate on the test tube bottom, abandon supernatant, adding waits the PBS mixing of sputum volume as the phlegm suspension in the sediment; With above-mentioned phlegm suspension the bacteria suspension of 1mg/ml being diluted to final concentration is 0.3 * 10
6Bar/ml contains bacterium phlegm dilution, gets the above-mentioned bacterium phlegm dilution 30 μ l that contain in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
D) preparation method of 4+ phlegm smear
Get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8PBS, centrifugal 3000g 30 minutes, makes in the phlegm solids concentrate on the test tube bottom, abandon supernatant, adding waits the PBS mixing of sputum volume as the phlegm suspension in the sediment; With above-mentioned phlegm suspension the bacteria suspension of 1mg/ml being diluted to final concentration is 2.25 * 10
6Bar/ml contains bacterium phlegm dilution, gets the above-mentioned bacterium phlegm dilution 30 μ l that contain in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
Embodiment 3 acid-fast bacillis derive from the preparation method of culture of bacteria bacterium colony
1, the preparation of bcg bacteria suspension
Get the bacterial clump (Shanghai Vaccine and Serum Institute provides) in two weeks of first growth of Bacille Calmette-Guerin and put into the 2ml centrifuge tube, wherein add the PBS that 4-5 beaded glass and 100 μ l contain 0.5% Tween-80, handle (80 ℃ of water-baths, 120 minutes) through bacteria inactivation; Behind the natural cooling, bacterium is ground in vibration, makes bacterium liquid be the cheese sample, after the PBS dilution that contains 0.5% Tween-80, with standard Maxwell opacity tube (MacFarland No.1) than turbid, be made into the bacteria suspension of 1mg/ml.
2, the preparation of positive spit smear
A) preparation method of 1+ phlegm smear
Get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 70 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8PBS, centrifugal 3000g 30 minutes, makes in the phlegm solids concentrate on the test tube bottom, abandon supernatant, adding waits the PBS mixing of sputum volume as the phlegm suspension in the sediment; With above-mentioned phlegm suspension the bacteria suspension of 1mg/ml being diluted to final concentration is 2 * 10
4Bar/ml contains bacterium phlegm dilution, gets the above-mentioned bacterium phlegm dilution 30 μ l that contain in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
B) preparation method of 2+ phlegm smear
Get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8PBS, centrifugal 3000g 30 minutes, makes in the phlegm solids concentrate on the test tube bottom, abandon supernatant, adding waits the PBS mixing of sputum volume as the phlegm suspension in the sediment; With above-mentioned phlegm suspension the bacteria suspension of 1mg/ml being diluted to final concentration is 2 * 10
5Bar/ml contains bacterium phlegm dilution, gets the above-mentioned bacterium phlegm dilution 30 μ l that contain in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
C) preparation method of 3+ phlegm smear
Get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8PBS, centrifugal 3000g 30 minutes, makes in the phlegm solids concentrate on the test tube bottom, abandon supernatant, adding waits the PBS mixing of sputum volume as the phlegm suspension in the sediment; With above-mentioned phlegm suspension the bacteria suspension of 1mg/ml being diluted to final concentration is 2 * 10
6Bar/ml contains bacterium phlegm dilution, gets the above-mentioned bacterium phlegm dilution 30 μ l that contain in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
D) preparation method of 4+ phlegm smear
Get the phlegm in morning (Shanghai international travel health care center provides) of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle (80 ℃ of water-baths, 30 minutes) through bacteria inactivation; Behind the natural cooling, contain in aseptic sputum is handled pipe, the NaOH-NALC mixing that adds 2 times of sputum volumes is after 20 minutes, be diluted to 50ml with PH6.8PBS, centrifugal 3000g 30 minutes, makes in the phlegm solids concentrate on the test tube bottom, abandon supernatant, adding waits the PBS mixing of sputum volume as the phlegm suspension in the sediment; With above-mentioned phlegm suspension the bacteria suspension of 1mg/ml being diluted to final concentration is 2.3 * 10
6Bar/ml contains bacterium phlegm dilution, gets the above-mentioned bacterium phlegm dilution 30 μ l that contain in the oval phlegm film of smearing on the microslide about 1 * 2cm, air dry, and flame is fixed.Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, be positioned in the sample box room temperature preservation.
2.3 testing result evaluation method
At present, acid-fast bacilli all adopts Ziehi-Neelsen stain in the laboratory detection sputum, finds during microscopy that the quantity of acid-fast bacilli is very important reference frame.Phlegm smear for microscopic examination result is not only qualitative to disease, also is the sxemiquantitative to disease severity.At first whether acid-fast bacilli exists in the judgement sample, if exist, acid-fast bacilli amount in average each visual field of report microscopically, the result represents (result reports that foundation is referring to [GB 15987-1995] infectiousness pulmonary tuberculosis diagnostic criteria and treatment principle) with " feminine gender ", " concrete bacterial population/300 visuals field ", " 1+ ", " 2+ ", " 3+ " and " 4+ ".
The expected results of test sample is determined when specimen preparation, after testing result is returned in each laboratory, determines the situation that meets of each test result of samples of laboratory and expected results.If testing result and expected results meet, then this sample detection result is 10 minutes; If testing result and expected results do not meet, we can check determining the accuracy of testing result test sample, and mark; Each sample top score is 10 minutes, each laboratory amount to 80 minutes and above for qualified, represent that this laboratory test item merit rating is qualified; The laboratory total is divided into defective less than 80, represent that this laboratory test item merit rating is defective; False negative result appears in " 1+ " above positive spit smear, is judged to be defective.
Testing result is estimated foundation referring to (1) GB 15987-1995 " infectiousness pulmonary tuberculosis diagnostic criteria and treatment principle "; (2) " phlegm smear for microscopic examination quality control (assurrance) manual " 2004, the CDC establishment.
2.4 the uniformity testing of sample
In order to guarantee the sample room there was no significant difference in every group, guarantee that the dissatisfied result who occurs in the ability verification is not attributed to the variation between the sample, guarantee the justice and the science of proficiency testing plan, the sample for preparing is carried out uniformity testing.From every group of 150 test sample, randomly draw 10 samples, sample is carried out acid-fast stain, respectively each sample is carried out microscopic examination, the quantity of observed acid-fast bacilli under numeration and 1-300 visual field of report by two specialized laboratories with Ziehi-Neelsen stain.Testing result is for judging the rank value (feminine gender, 1+, 2+, 3+, 4+) that obtains according to each sample acid-fast bacilli amount.Testing result and expected results are compared.Negative sample group (see figure 5): 10 sampling sample result are all negative, illustrate that then this group sample is even; 1+ sample sets (see figure 4): 10 sampling sample result are 1+ or 2+ or 1-8 bar/300 visuals field, illustrate that then this group sample is even; 2+ sample sets (see figure 3): 10 sampling sample result are 1+ or 2+ or 3+, illustrate that then this group sample is even; 3+ sample sets (see figure 2): 10 sampling sample result are 2+ or 3+ or 4+, illustrate that then this group sample is even; 4+ sample sets (see figure 1): 10 sampling sample result are 3+ or 4+, illustrate that then this group sample is even.Negative findings appears in " 1+ " above phlegm smear, illustrates that population sampled is inhomogeneous.
This ability verification has designed 1-10 group sample, every group of 150 phlegm smears.Randomly draw 10 smears and detect from each group, the result is consistent with expected results.So the sample homogeneity of this preparation meets the requirements.
2.5 stability of sample check
For the reliability of check sample in transportation, stability in the storage and packing, means of transportation, guarantee that sample meets the requirements from being prepared into the stability that detects the whole process, prepared sample is carried out stability test before the proficiency testing sample is provided and proficiency testing implement stability test in the overall process.
The stability test of phlegm smear 2.5.1 be unstained: randomly drawing needs the phlegm of quantity smear, and according to being delivered to laboratory, different regions (home or overseas) soon after the above-mentioned manner of packing packing, room temperature preservation is returned in the express delivery mode to planning Return Date.Carry out the acid-fast bacilli detection with Ziehi-Neelsen stain to returning the phlegm smear.The result and the expected results that detect are compared, and negative phlegm smear: testing result is all negative, and phlegm smear qualified stability then is described; 1+ phlegm smear: testing result is 1+ or 2+ or 1-8 bar/300 visuals field, and phlegm smear qualified stability then is described; 2+ phlegm smear: testing result is 1+ or 2+ or 3+, and phlegm smear qualified stability is described; 3+ phlegm smear: testing result is 2+ or 3+ or 4+, and phlegm smear qualified stability is described; 4+ phlegm smear: testing result is 3+ or 4+, and phlegm smear qualified stability is described; Negative findings appears in " 1+ " above phlegm smear, and phlegm smear instability is described; If the result is variant, phlegm smear instability is described, need search the unsettled reason of phlegm smear, prepare the phlegm smear again or change storage, means of transportation.
2.5.2 the stability test of dyeing phlegm smear: randomly drawing needs the phlegm of quantity smear, carries out being delivered to laboratory, different regions (home or overseas) soon after the acid-fast stain of phlegm smear with Ziehi-Neelsen stain, and room temperature preservation is returned in the express delivery mode to planning Return Date.Search acid-fast bacilli with microscopic method, result and the expected results that detects compared, negative phlegm smear: testing result is negative, and phlegm smear qualified stability is described; 1+ phlegm smear: testing result is 1+ or 2+ or 1-8 bar/300 visuals field, and phlegm smear qualified stability is described; 2+ phlegm smear: testing result is 1+ or 2+ or 3+, and phlegm smear qualified stability is described; 3+ phlegm smear: testing result is 2+ or 3+ or 4+, and phlegm smear qualified stability is described; 4+ phlegm smear: testing result is 3+ or 4+, and phlegm smear qualified stability is described; Negative findings appears in " 1+ " above phlegm smear, and phlegm smear instability is described; If the result is variant, phlegm smear instability is described, need search the unsettled reason of phlegm smear, prepare the phlegm smear again or change storage, means of transportation.
The testing result of be unstained sample and stained specimens that two areas that this ability verification is chosen come and go is consistent with expected results, can think that sample is stable, meets the requirement of this proficiency testing plan.
2.6 the sign of sample
Computer programming designs one group of random sequence number that contains the 1-2000 numeral and do not have the repetition numeral.According to 150 numerals is one group, has selected 10 groups of numerals altogether, corresponding to every group of sample, makes each sample obtain unique Digital ID the numeral in every group.In the Digital ID of this proficiency testing sample the 1st to 4 for expression area, Shanghai, the 5th, 6 bit representation preparation times, be sample sequence number since the 7th bit digital.
2.7 the packing of sample and transmission
Consider transportation safety and stability of sample, test sample all adopts three layers of packing: ground floor is placed on sample (phlegm smear) in the special-purpose Glass carrier box, and the second layer is at Glass carrier box external application antidetonation barrier film parcel, and the 3rd layer is external application waterproof paper case packing.Also have ability verification explanation, sample reception state confirmation table and test results report table with what sample together sent.
Whether before sample sends, it is intact to examine sample state by the special messenger, recording laboratory code and corresponding sample number.Participate in the experiment after the chamber receives sample, fill in sample reception state confirmation table, signature back fax or post back this proficiency testing contact person is intact with the state of affirmation test sample.
If the situation that influence such as sample breakage detects requires to get in touch this proficiency testing contact person as early as possible, so that reissue test sample.
2.8 the recovery of sample
In order to estimate the detectability of each chamber of participating in the experiment accurately, equitably, guarantee proficiency testing result's reliability, require test sample is together returned with report as a result in the detection back that finishes.
Participate in the experiment test sample that the chamber returns by carrying out outcome evaluation with expected results comparison and expert's audit mode, the accurate and just of result can be guaranteed on the one hand, respectively the participate in the experiment problem of chamber testing result existence and the standardized degree of part operation can be found on the other hand.
2.9 testing result statistical method
Phlegm smear Ziehi-Neelsen stain is searched the judgement of the testing result of acid-fast bacilli and is taked qualitative in conjunction with semiquantitative mode.Detect the expected results of phlegm smear and when it prepares, determined, after tested laboratory or the individual reports's testing result, determine the testing result of each phlegm smear of laboratory and the situation that meets of expected results.
Negative phlegm smear: testing result is negative, illustrates that testing result and expected results meet; 1+ phlegm smear: testing result is 1+ or 2+ or 1-8 bar/300 visuals field, illustrates that testing result and expected results meet; 2+ phlegm smear: testing result is 1+ or 2+ or 3+, illustrates that testing result and expected results meet; 3+ phlegm smear: testing result is 2+ or 3+ or 4+, illustrates that testing result and expected results meet; 4+ phlegm smear: testing result is 3+ or 4+, illustrates that testing result and expected results meet; Negative findings appears in " 1+ " above phlegm smear, illustrates that testing result and expected results do not meet.
Testing result is estimated foundation referring to (1) GB 15987-1995 " infectiousness pulmonary tuberculosis diagnostic criteria and treatment principle "; (2) " phlegm smear for microscopic examination quality control (assurrance) manual " 2004, the CDC establishment.
3 laboratory detection result and evaluation thereof
3.1 testing result gathers
This proficiency testing is taked qualitative in conjunction with semiquantitative mode to the judgement of testing result.Each chamber of participating in the experiment is received 10 test sample altogether.The chamber of participating in the experiment should conform to expected results or the actual result who checks to the result of determination of 10 samples, and integrate score 80 minutes and when above, and it is qualified that the detectability of this project is judged to be, otherwise be judged to be defective; False negative appears in " 1+ " above positive spit smear, is judged to be defective.Test result of samples that this proficiency testing has had 43 tame laboratory reports.
3.2 evaluation of result
In repayment result's 43 tame laboratories, there are 30 tame laboratories to obtain and get a mark of 100,10 tame laboratories obtain 95 fens, and 1 tame laboratory obtains 90 fens, and 2 tame laboratories obtain 85 fens.The chamber of the participating in the experiment scoring of 43 families is all more than 80 minutes, and is all qualified.Have the situation that do not meet that low false positive (4) and quantization error (12) appear in 16 testing results, relate to 13 tame laboratories.
This proficiency testing sends 440 test sample altogether, has repaid 430 test result of samples, has 414 sample detection results correct, accounts for 96.3% of repayment sample number.
4 technical Analysis and suggestion
4.1 the quality control of testing process
The acid-fast bacilli proficiency testing is to the quality control requirement height of testing process in the smear staining method detection sputum.Dust can cause the pollution of sample in environment, especially water quality and the air, safeguards that as the microscope that uses improper meeting influence testing result in the detection.So the laboratory must be noted detecting water and clean environment, strictness prevents operational pollution, and this is to guarantee the correct basis of testing result.
4.2 detection method and reagent
The detection method of this proficiency testing project relates to phlegm smear Ziehi-Neelsen stain and microscope inspection survey technology, and the quality of the acid-fast stain reagent of Ziehi-Neelsen stain and use is closely related.In the 43 tame laboratories of participating in, there are 38 tame laboratories to use the acid-fast stain reagent of same brand, there are 2 families to use the acid-fast stain reagent of different brands, there are 3 tame laboratories not report the brand of used detectable.This time do not find the appreciable impact of reagent in the ability verification to testing result.
5 sum up
In this proficiency testing project a plurality of innovative points are arranged.At first, it is the most basic, a most widely used project in the test item of tuberculosis laboratory that the acid-fast bacilli test item is searched in the acid-fast stain of phlegm smear, but the laboratory proficiency testing project that similar project undertaken by a plurality of different bacterium content, the multiple test sample combination of identical bacteria content sample like this is at home still first, and the proficiency testing of the microorganism of medical laboratory specialty morphologic detection project to be implemented in domestic also be to carry out first.
Secondly, in proficiency testing was implemented, we had adopted the result treatment mode that test sample is reclaimed, and the result who causes with eliminating sample reason does not meet, and has as much as possible guaranteed result's the accuracy and the fairness of implementing plan, and workable.In addition, the method for sample homogeneity check and stability test also is to use on the morphologic proficiency testing project of medical science first in the checking of this ability, from statistics, has all satisfied the effect of test sample being carried out homogeneity and stability assessment.
Claims (13)
1. a phlegm smear that is used for the microexamination of ability verification acid-fast bacilli is characterized in that, it comprises feminine gender, 1+, 2+, 3+, the 4+ phlegm smear of totally five kinds of acid-fast bacilli content, and the acid-fast bacilli content of this feminine gender phlegm smear is 0/300 visuals field; The acid-fast bacilli content of this 1+ phlegm smear is 3-9 bar acid-fast bacilli/100 visuals field; The acid-fast bacilli content of this 2+ phlegm smear is 1-9 bar acid-fast bacilli/10 visuals field; The acid-fast bacilli content of this 3+ phlegm smear is a 1-9 bar acid-fast bacilli/visual field; The acid-fast bacilli content of this 4+ phlegm smear is＞9 an acid-fast bacillis/visual field; Described acid-fast bacilli derives from sputum or culture of bacteria bacterium colony.
2. the preparation method of a phlegm smear as claimed in claim 1 is characterized in that, the acid-fast bacilli that described phlegm smear contains derives from sputum, and this method comprises the steps:
1) get the phlegm in morning of different tuberculosis patients, be positioned in the aseptic sputum specimen collection container, bacteria inactivation is handled;
2) behind the natural cooling, contain in aseptic sputum is handled pipe, make sputum liquefaction and with after the PBS dilution, centrifugal, make bacterium concentrate on the test tube bottom, abandon supernatant, add the PBS mixing in the sediment as the phlegm suspension, as initial grade concentration, calculate extension rate according to acid-fast bacilli purpose grade concentration with phlegm smear acid-fast stain sediments microscope inspection method bacterial detection quantity then, and be diluted to the phlegm dilution of purpose grade concentration with PBS;
3) get above-mentioned phlegm dilution and on microslide, smear ovalization phlegm film, air dry, flame is fixed; Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, room temperature preservation.
3. the preparation method of phlegm smear as claimed in claim 2 is characterized in that, in step 2) in, the purpose grade concentration of acid-fast bacilli is 0/ml in the described phlegm dilution, 0.8-2.2 * 10
4Bar/ml, 0.3-2.2 * 10
5Bar/ml, 0.3-2.2 * 10
6Bar/ml, perhaps＞2.2 * 10
6Bar/ml.
4. the preparation method of phlegm smear as claimed in claim 2, it is characterized in that, after step 3) is finished, also comprise following packaging step: the phlegm smear adopts three layers of packing, ground floor is placed on the phlegm smear in the special-purpose Glass carrier box, the second layer is at Glass carrier box external application antidetonation barrier film parcel, and the 3rd layer is external application waterproof paper case packing.
5. the preparation method of phlegm smear as claimed in claim 2, it is characterized in that, in the step 3), described sample number into spectrum is that computer programming designs one group of random sequence number that does not have the repetition numeral, should numeral corresponding to each phlegm smear, make each sample obtain unique Digital ID, numeral preparation area, preparation time and phlegm smear sequence number in this Digital ID.
6. the preparation method of a phlegm smear as claimed in claim 1 is characterized in that, the acid-fast bacilli that described phlegm smear contains derives from the culture of bacteria bacterium colony, and this method comprises the steps:
1) preparation of bcg bacteria suspension: the bacterial clump of getting two weeks of first growth of Bacille Calmette-Guerin is put into glass bacteria grinder bottom or is had the centrifuge tube of beaded glass, wherein adds the PBS that contains 0.5% Tween-80, handles through bacteria inactivation; Behind the natural cooling, fully stir vibration and make bacterium liquid be the cheese sample, with physiological saline or PBS mill bacterium and the dilution that contains 0.5% Tween-80, with standard Maxwell opacity tube than turbid, be made into the bacteria suspension of 1mg/ml;
2) get the phlegm in morning of different non-tuberculosis patients, be positioned in the aseptic sputum specimen collection container, handle through bacteria inactivation;
3) behind the natural cooling, contain in aseptic sputum is handled pipe, make sputum liquefaction and with the PBS dilution, centrifugal, make that solids concentrates on the test tube bottom in the phlegm, abandon supernatant, the PBS mixing of sputum such as addings volume is as the phlegm suspension in the sediment;
4) use phlegm smear acid-fast stain sediments microscope inspection method bacterial detection quantity as initial grade concentration, calculate extension rate according to acid-fast bacilli purpose grade concentration then, what the bacteria suspension that the phlegm suspension made from step 3) is made step 1) was diluted to purpose grade concentration contains bacterium phlegm dilution; Get the above-mentioned bacterium phlegm dilution that contains and on microslide, smear ovalization phlegm film, air dry, flame is fixed; Paste anticorrosion slip-off preventing label at microslide one end 1/3 place and indicate sample number into spectrum, room temperature preservation.
7. the preparation method of phlegm smear as claimed in claim 6 is characterized in that, in step 4), the described purpose grade concentration that contains acid-fast bacilli in the bacterium phlegm dilution is 0/ml, 0.8-2.2 * 10
4Bar/ml, 0.3-2.2 * 10
5Bar/ml, 0.3-2.2 * 10
6Bar/ml, perhaps＞2.2 * 10
6Bar/ml.
8. the preparation method of phlegm smear as claimed in claim 6, it is characterized in that, after step 4) is finished, also comprise following packaging step: the phlegm smear adopts three layers of packing, ground floor is placed on the phlegm smear in the special-purpose Glass carrier box, the second layer is at Glass carrier box external application antidetonation barrier film parcel, and the 3rd layer is external application waterproof paper case packing.
9. the preparation method of phlegm smear as claimed in claim 6, it is characterized in that, in the step 4), described sample number into spectrum is that computer programming designs one group of random sequence number that does not have the repetition numeral, should numeral corresponding to each phlegm smear, make each sample obtain unique Digital ID, numeral preparation area, preparation time and phlegm smear sequence number in this Digital ID.
10. the uniformity detecting method of a phlegm smear as claimed in claim 1 is characterized in that, comprises the steps:
1) from the phlegm smear of same levels concentration, randomly draws and need the phlegm of quantity smear, with Ziehi-Neelsen stain the phlegm smear of sampling is carried out acid-fast stain, and each the phlegm smear that extracts carried out microscopic examination, the quantity of observed acid-fast bacilli under numeration and 1-300 visual field of report;
2) testing result is for judging the rank value that obtains according to the acid-fast bacilli amount in each phlegm smear: feminine gender, 1+, 2+, 3+, 4+; Testing result and expected results are compared; Negative phlegm smear: sampling phlegm smear results is all negative, illustrates that then population sampled is even; 1+ phlegm smear: sampling phlegm smear results is 1+ or 2+ or 1-8 bar/300 visuals field, illustrates that then population sampled is even; 2+ phlegm smear: sampling phlegm smear results is 1+ or 2+ or 3+, illustrates that then population sampled is even; 3+ phlegm smear: sampling phlegm smear results is 2+ or 3+ or 4+, illustrates that then population sampled is even; 4+ phlegm smear: sampling phlegm smear results is 3+ or 4+, illustrates that then population sampled is even; Negative findings appears in " 1+ " above phlegm smear, illustrates that population sampled is inhomogeneous.
11. the Detection of Stability method of a phlegm smear as claimed in claim 1 is characterized in that, comprises the steps:
1) randomly draw and need the phlegm of quantity smear, be delivered to the laboratory, different regions after the packing soon, room temperature preservation is returned in the express delivery mode to planning Return Date, to after returning the phlegm smear and carrying out acid-fast stain, searches acid-fast bacilli with microscopic method with Ziehi-Neelsen stain; Perhaps randomly drawing needs the phlegm of quantity smear, carries out being delivered to the laboratory, different regions soon after the acid-fast stain of phlegm smear with Ziehi-Neelsen stain, and room temperature preservation is returned in the express delivery mode to planning Return Date, searches acid-fast bacilli with microscopic method;
2) result that will detect and expected results compare, and negative phlegm smear: testing result is all negative, and phlegm smear qualified stability then is described; 1+ phlegm smear: testing result is 1+ or 2+ or 1-8 bar/300 visuals field, and phlegm smear qualified stability then is described; 2+ phlegm smear: testing result is 1+ or 2+ or 3+, and phlegm smear qualified stability is described; 3+ phlegm smear: testing result is 2+ or 3+ or 4+, and phlegm smear qualified stability is described; 4+ phlegm smear: testing result is 3+ or 4+, and phlegm smear qualified stability is described; Negative findings appears in " 1+ " above phlegm smear, and phlegm smear instability is described; If the result is variant, phlegm smear instability is described, need search the unsettled reason of phlegm smear, prepare the phlegm smear again or change storage, means of transportation.
12. the application of a phlegm smear as claimed in claim 1 in proficiency testing.
13. a phlegm smear as claimed in claim 1 is the application in quality evaluation and the indoor quality control between the chamber, laboratory.
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| CN103822815A (en) * | 2014-03-25 | 2014-05-28 | 吉安协和医院 | Method for inspecting acid-fast bacilli by liquid-based thin-layer cell smears |
| CN108587973A (en) * | 2018-05-15 | 2018-09-28 | 首都医科大学附属北京胸科医院 | A kind of manual simulation's phlegm and the preparation method and application thereof |
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| CN108587973A (en) * | 2018-05-15 | 2018-09-28 | 首都医科大学附属北京胸科医院 | A kind of manual simulation's phlegm and the preparation method and application thereof |
| CN111309781A (en) * | 2020-01-21 | 2020-06-19 | 杭州杏林信息科技有限公司 | Method and equipment for counting number of pathogen censored persons before treatment by antibacterial drugs |
| CN111309781B (en) * | 2020-01-21 | 2023-04-18 | 杭州杏林信息科技有限公司 | Method and equipment for counting number of pathogen censored persons before treatment by antibacterial drugs |
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| CN112481402A (en) * | 2020-12-29 | 2021-03-12 | 上海国际旅行卫生保健中心(上海海关口岸门诊部) | Primer group for M.tuberculosis MLST typing detection based on Sanger sequencing and application thereof |
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