说明成果ral培训training stainer-09

1、RAL stainer AS training1Chapter 1: Introduction What is tuberculosis (TB)? World overview TB diagnosis TB : Which type of bacteria is it?3WHAT IS TUBERCULOSIS (TB) ?Pathology: Tuberculosis disease is caused by a bacterium that can affect any part of the body, but most commonly the lungs. 4WHAT IS TU

2、BERCULOSIS (TB) ?Pathology: Only TB of the lung or throat can be infectiousand is generally transmitted by prolonged and/or frequent contact with an infected individual who is coughing bacteria droplets into the surrounding air. Bacteria are breathed in through the lungs,can travel in the blood to o

3、ther organs which e infected. 5World overview (WHO information)WHO (World Health Organization) estimation : around 2 billion people are infected with Mycobacterium tuberculosis. .TB2 billion = one-third of the worlds population6World overview (WHO information)In 2012 : estimated 8.6 million new case

4、s of TB (13% co-infected with HIV) 1.3 million people died from TB, 320 000 people died of HIV-associated TB. Almost 25% of deaths among people with HIV are due to TB1.1 million new cases of HIV-positive new TB casesPrevalence - HBC/LBC high-burden country = In 2012, about 80% of reported TB cases o

5、ccurred in 22 countries.7World overview (WHO information)Prevalence - HBC/LBCMost of the estimated number of cases in 2011 occurred in Asia (59%) Africa (26%);Eastern Mediterranean Region (7.7%), European Region (4.3%)Region of the Americas (3%).8TB DIAGNOSIS Symptoms : Most of the TB cases (among a

6、dults aged 15 to 59 years) are curable disease, so diagnosis is key.Fever, Tirednesscough more than three weeks : cough,dyspnea, sputum, hemoptysis, and / or general symptomsnight sweats, fever,weight loss,.9TB DIAGNOSIS Steps :- perform a lungs radiograph (Chest X-Ray)- Diagnosis of certitude is mi

7、crobiological10TB : Which type of bacteria is it?Cell wall structure : different from each other11TB : Which type of bacteria is it?Contains : peptidoglycan, complex lipids : over 60% Cell wall structure12TB : Which type of bacteria is it?High concentration of lipids in the cell wall : Impermeabilit

8、y to stains and dyes Resistance to many antibiotics Resistance to killing by acidic and alkaline compounds Resistance to osmotic lysis via complement deposition Resistance to lethal oxidations and survival inside of macrophages TB is called Acid-Fast Bacilli (AFB) Bacilli Acido Alcoolo Resistant (B.

9、A.A.R.) in EuropeSlow growth : culture during 42 days on agar medium 14 days in liquid medium 13TB : Which type of bacteria is it?This wall structure (lipidic) makes inefficient usual stains. It is necessary to use specific technics to stain it.14Chapter 2: WorkflowTB SAMPLEDECONTAMINATION & FLUIDIF

10、ICATIONFIXATION (heat & chemical)STAININGWORKFLOW Where does our offer stand in he TB detection process Preparation15CollectionMicroscopicexaminationcultureID/ASTUsual workflowTB workflowPREVI Fluo TB / RAL stainer (main business)CollectionFixationStainingReadingPositive cultureStainingID / ASTPREVI

11、 Fluo TB / RAL stainer (secondary business)MicroscopicexaminationSampleSamples : Mainly sputum (80%) & expectorations but also : urines, bones, liquids,biopsy.Collection must be done on 3 consecutive days Use sterile vial collectionCollectionPreparationStainingStainingFixationPositive cultureReading

12、ID/AST16SampleSamples : stored and shipped in +4C conditions until it transfer to the lab, to avoid the bacilli and bacteria start growing and modify the diagnostic. A bad storage could modify the culture interpretation.CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST17Prep

13、aration for direct exam Safety :Due to the mode of transport of the bacilla(particles of water in the air), all the sample manipulation should be made in a safety cabinet supposed to be located in a BSL3.CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST18(BioSafety Level 3)P

14、reparation for direct exam CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST19DECONTAMINATIONFLUIDIZATION2 steps to be adapted to the local contextPreparation for direct exam : Decontamination Goal : Avoid any other bacteria to interfere in the culture process Challenge : Ki

15、ll human commensal bacteria and not mycobacteria. Decontamination has no impact on the stainingBut you cannot grow cultures without decontaminationCollectionPreparationStainingStainingFixationPositive cultureReadingID/AST20First thing to do is decontamination : Preparation for direct exam : Fluidiza

16、tionGoal :Release mycobacteria embedded in the sputum to get better staining results.CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST21Second thing to do is fluidizationMain technique used worldwide is called Kubica : to decontaminate : Sodium Hydroxide (NaOH). to neutraliz

17、e NaOH : Buffer (allow mycobacteria to grow in culture)- to fluidificate the sputum N-Acetyl L-Cysteine (NALC)CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST22Preparation for direct exam: Decontamination & FluidizationMost of the commercial kits perform both fluidification

18、 and decontamination in one step.Possible to stain without fluidization mended as it improves resultsNext step requested :centrifugation 3000 trs/min during 20 to 30 minutes Microscopic examination (staining) is performed on the pellet.CollectionPreparationStainingStainingFixationPositive cultureRea

19、dingID/AST23Preparation for direct exam: Decontamination & FluidizationPreparation: smearing step Goal : make the smear as thin as possibleMethod : Use a loop to spread the sample on the slideSpread : movement continuous rotationCollectionPreparationStainingStainingFixationPositive cultureReadingID/

20、AST24Preparation: smearing step Method : A drop from the pellet on a slide, dry on a heating blockUse a slide to smear the sample on another slide. CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST25TB : Fixation of the slideCollectionPreparationStainingStainingFixationPosit

21、ive cultureReadingID/AST26Fixation step must be performed: Heat fixation Goal : avoid cross contaminationprepare the bacilli barrier to be penetrated by the stain without serious distortion (cell morphology protection)Principle: make the smear adhere to the slideFixation of a direct exam: heat fixat

22、ionHeat fixationGoal: stick the smear to the slide by removing as much water as possible from the sample (let dry the slides under a safety cabinet from 15 to 30 minutes) limit the pathogenic characteristics of the positive smear (kill as much mycobacteria as possible)This “external” fixation allows

23、 to preserve the bacteria morphology CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST27Fixation of a direct exam: heat fixationHeat fixationMethod : 10 minutes at 70C on a heating block (minimum)Add before few drops of alcohol : Fast evaporation fluids are very efficient to

24、 extract water from the smearThe cumulative effect of heat and evaporation increase the efficiency of the process.CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST28Fixation of a direct exam: heat fixationHeat fixationMethod : Then fix slides under the blue flame of bunsen b

25、urner briefly smear turn upwardsCollectionPreparationStainingStainingFixationPositive cultureReadingID/AST29TB : STAININGCollectionPreparationStainingStainingFixationPositive cultureReadingID/AST30High concentration in lipid (wall structure) in mycobacteria makes inefficient usual stains.It is neces

26、sary to use specific technics to stain it2 manual staining methods : Covering the slide with stainsDipping the slide into wellsSTAINING : WHO mendationsCollectionPreparationStainingStainingFixationPositive cultureReadingID/AST31Before 1996: Ziehl Nielsen + usual microscope (white light)From 1996: 1s

27、t mention of fluorescence as an help for screening. ZN is still the reference method.From 2008: Fluorescence is mentioned as an IMPROVEMENT to ZN techniqueIn 2009, the red counter stain is mentioned for the 1st time as a good alternative to dark background.2010: Fluorescence is the reference method.

28、STAINING : Hot/Cold Ziehl-Neelsen CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST32PRINCIPLE :Three stages : applying a strong stain hot or coldsuccessive discoloration with a strong acid and then with alcohol 90one counter stainZNUsed to be reference technique Less used :

29、 risks for manipulatorfuchsin is highly toxic and carcinogenicespecially when hot due to fumesSTAINING : AURAMINE (described by Degommier)CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST33PRINCIPLE :Auramine is used as fluorochromeAfter treatment with acid and alcohol, myco

30、bacteria keep the auramine stainingThiazine red is used to counter stain the other bacteria (non acido-alcoolo-resistant)AURAMINE more and more used2010 : WHO mentioned it as reference methodREADINGCollectionPreparationStainingStainingFixationPositive cultureReadingID/AST34Depending on the staining,

31、 reading is performed under - normal microscope (white light) : ZN- fluorescence microscope : AURAMINEREADING : Ziehl Neelsenexamined at least 100 fields (very long)magnification of x100three scans of the smear should be performedCollectionPreparationStainingStainingFixationPositive cultureReadingID

32、/AST35Mycobacterium tuberculosisx100READING : AuramineCollectionPreparationStainingStainingFixationPositive cultureReadingID/AST36Mycobacterium tuberculosisx40examined at least 30 fields magnification of x 40Mycobacteria appear fluorescent yellow on a red backgroundSlides need to be read as soon as

33、possible after the staining Slide require absolute protection from lightIn parallel of the microscopic examination you have to culture (from the pellet)Culture is confirmation of the staining reading result (42 days)- Lowenstein-Jensen medium (ref 42089) reference medium mended by the IUATLD (Intern

34、ational Union Against Tuberculosis and Lung Disease)37CULTURE- Coletsos medium (ref 42082)CollectionPreparationStainingStainingFixationPositive cultureReadingID/ASTPREVI Fluo TB / RAL stainer PREVI Fluo TB / RAL stainerWhere does our offer stand in the TB detection process ?CollectionPreparationStai

35、ningStainingFixationPositive cultureReadingID/AST3839Chapter 3 : ReagentsFLUO RAL COLD ZN40RAL STAINER : focus on reagents Flexibility FLUO RAL COLD ZN Use of different kits with the same instrument No rinsing or cleaning between use of different kits One kit at a timeChange kit in 2 minutesOR41RAL

36、STAINER : focus on reagents Fluo RALFLUO RAL (Auramine)Storage temperature : 15-25C away from light 1- Fixative Fluo-RAL2- Auramine Fluo-RAL -Solution for Auramine 3- Discolouring solution Fluo-RAL 4- Thiazine Red Fluo-RAL - Solution for Thiazine Red Number of tests possible : around 300 slides (=30

37、 cycles) and/or 1 week of use after opening. 42RAL STAINER : focus on reagents Fluo RALFLUO RAL (Auramine)Solutions preparation : Auramine solution bottle 2 : Add the content of “Solution for Auramine” in the bottle 2 “Auramine Fluo-RAL”Thiazine Red solution bottle 4 : Add the content of “Solution f

38、or Thiazine Red” in the bottle 4 “Thiazine Red Fluo-RAL”ReconstitutionFluo RAL : fixative solutionChemical fixation : agent penetrating bacteria prepare the internal structure of cells for stainingavoid cross-contaminationhas been developed specifically for mycobacteriaFixation solution (bath 1)Acid

39、 trichloroacetic43TB : Fixation of a direct examDoes not replace heat fixation !BOTH fixation are mandatory to work with the RAL STAINERA good staining is also due to a good fixation !CollectionPreparationStainingStainingFixationPositive cultureReadingID/AST44The slide is still potentially pathogen.

40、 No study has been conducted to prove that the slides are 100% inactivated by this process. Each lab is managing its own safety. 45RAL STAINER : focus on reagents Fluo RALFLUO RAL (Auramine)ReadingRequires a Fluorescent microscope. Screening at x 20 Confirmation at x 40 only in few cases5 minutes /

41、slide (reading time) 5 to 6 times quickerImproved sensitivity No fluorescent artifact Better contrast (green fluorescence on red background)Mycobacterium tuberculosisx20 - screeningx40- confirmation46RAL STAINER : focus on reagents Fluo RAL47RAL STAINER : focus on reagents Fluo RAL48RAL STAINER : fo

42、cus on reagents Fluo RALRAL STAINER : focus on reagents Fluo RALThe red background :Help the reader to stay focused Very helpful for non experienced usersEven with a good digestion, bacilla are usually concentrated in red parts of the smear.The intensity of the mycobacteria stained by auramine :The

43、Green yellowish is bright and flashy 49AdvantagesRAL STAINER : focus on reagents Fluo RALSpecificity of the staining :No artefacts Quality of the fixative solution :Allows dipping processes Stability of the solutions prior mixing: The auramine and the thiazine red have been separated from their carb

44、olic component to improve the stability and the resistance to thermal shock in shipment/storage.50Advantages51RAL STAINER : focus on reagents COLD ZNCOLD ZN1- Fixative solution 2- Fuchsin 3- Discolouring solution 4- Methylene blue Number of tests possible: 500 slides (=50 cycles) and/or 2 weeks of u

45、se after opening Storage temperature : 15-25 C away from light 52RAL STAINER : focus on reagents COLD ZNCOLD ZNClassic microscope (white light)Screening at x 50 Confirmation at x 100 always required30 minutes / slide (reading time)Reading53RAL STAINER : focus on reagents COLD ZNRAL STAINER : Ziehl N

46、eelsen versus AuramineZiehl Neelsen is still used in several occasions :To have information of the species of mycobacteria (tuberculosis, bovis, africae, .) by giving some detailsTo confirm positive cultureSometimes to confirm positive fluorescence54Ziehl Neelsen : confirmation of fluorescence ?Myco

47、bacterium tuberculosisHot Ziehl Neelsen staining x100Auramine x4055Chapter 4 : RAL TB STAINEROverviewTechnical advantages Instrument installationOn the instrument:User registrationSet up reagentsStaining protocolRun a stainingMicroscopy56Instrument overview : RAL TB STAINER57Instrument overview : RA

48、L TB STAINERTouch screen interfaceStandby buttonProtective coverStaining armStaining stationWith sealed lid(x4)Slide loading/unloadingDrawers and drying (x2)Draining nozzleRinsing zone 58Technical advantagesChoice of a well known mechanical system : Rotating handleReliability of the stainingConsiste

49、ncy of the stainingNo mechanical part in contact with the stains Level detection for each bath : the whole smear is always stainedEach bath is opened at the very last moment, just before dipping the slides in itLimit evaporation and limit the emission of fumes, possibly hazardousBath59Technical adva

50、ntagesThe air inside the machine is filtered with carbon filterNo maintenance = No cleaning process with any hazardous substance Rinse and used stains are automatically evacuated to a level controlled wasteUsers cant open the stainer during the process : No risk with mechanical part in movementSafe6

51、0Technical advantagesRFID SYSTEM (Radio Frequency Identification) SECURITY Stains loaded are automatically recognized : No risk of mis-usingThanks to RFID, the stainer recognizes automatically the type of kit loadedHelp the management of stains : As soon as the maximum number of cycles is reached, i

52、t suggests to change the stain kittraceability : Through a USB port, the user is able to collect a huge number of information: dates, procedures, number of cycles realized, number of cycles available, batch numbers, expiry dates, date of first use, usersUsers/Rights Management : A management of user

53、s by password and different levels of rights are adjustable in the system61INSTRUMENT INSTALLATION : UNPACKINGWooden box characteristics : Size : W 810 x D 525 x H 840Gross weight : 56,2 kg (weight of the cover alone : 12,8 kg)Instrument net weight : 28 kg62INSTRUMENT INSTALLATION : UNPACKINGUnlock

54、latches (4)Take the cover by the two handles on the sides (one or two people) then lift it up completely to be able to discover the equipment.63INSTRUMENT INSTALLATION : UNPACKINGRemove - the polystyrene protection around the equipment - the plastic bag covering the Stainer - the box containing acce

55、ssories 64INSTRUMENT INSTALLATION : UNPACKINGRemove the Stainer from the pallet :2 metallic rods maintain the Stainer fastened on the pallet. Use a 10mm wrench to unscrew nuts.65INSTRUMENT INSTALLATION : UNPACKINGSlightly lift up the RAL Stainer to be able to remove both metallic rods.When 2 rods ar

56、e removed, you can then take the RAL Stainer and place it on a stable, flat and clean table.Open the protective cover (press the locking finger behind the screen and turn the cover) and remove the blocking foam of the arm. 66INSTRUMENT INSTALLATION : UNPACKINGWe mend placing back both metallic rods

57、on the pallet to keep all elements necessary to repack the instrument.67INSTRUMENT INSTALLATION : UNPACKING68INSTRUMENT INSTALLATION : UNPACKINGThe RAL STAINER REFERENCE 414195 contains the instrument and all the starting kit (stylus, tanks, level detector, slide holders, fan filter, USB stick memor

58、y)The cord for the main supply is a Europe one, purchase locally the plug. The “installation kit” , ref 415735, needs to be order separately69INSTRUMENT INSTALLATION : UNPACKINGThe packaging contains the following items :70INSTRUMENT INSTALLATION : ENVIRONNEMENTThe appliance must be placed on a stab

59、le, flat and clean table. Avoid shocks and vibrations. Considering the weight of the RAL Stainer (28 kg), use a sufficiently solid table.Place the appliance close to a main power outlet and a running water tap if you have selected the running water connection option. Considering the size of the RAL

60、Stainer ( 42.5 cm / H 62.4 cm), plan to use a space of 70 x 70 cm for the appliance installation, use and maintenance. 71INSTRUMENT INSTALLATION Connect the supply cable to the apparatus at the place provided (see diagram), and to a nearby mains outlet.72INSTRUMENT INSTALLATION Rinsing liquid contai