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1. Note This batch of Auramine O is bad all has been discarded Hydrochloric acid is OK Batches checked on 18 5 08 Au O batch 18 5 08 Potassium permanganate batch 18 5 08 old Hydrochloric acid solution Average grading positive controls LED 40 x objective no 364 151 40 fields no 428 147 40 fields Control Slide AFB color AFB number Decolorization Decision 364 1 Bright yellow 124 40 40x F OK Accept Au O 428 1 Bright yellow 120 40 40x F OK Accept Au O NEG Bright yellow 15 40 40x F OK Reject others NEG NA None in 40 40x F OK Accept others Note contamination probably Auramine O To be checked further using only one of the new staining reagents on negative controls old good stains of the other types Module 6 Preparation of FM Reagents Page 9 of 11 Blank Log Book Sheet Quality Control of Staining Reagents This sheet can be reproduced for individual laboratory use Auramine O stains Batches checked on date Au O batch Hydrochloric acid batch Potassium permanganate batch Average grading positive controls LED 40 x objective no 40 fields no 40 fields Control slide AFB color AFB number Decolorization Decision Note Batches checked on Au O batch Hydrochloric acid batch Potassium permanganate batch Methylene blue batch if used Average grad
2. As per LQAS According to National TB Guidelines Page 4 of 15 Onsite evaluation Monitoring visits will be performed by the Supervisory Site and frequency will vary according to the study phase see Table 1 above During some of these visits the laboratory will receive an onsite evaluation OSE by the supervisor Table 1 These visits allow the worker to be observed under actual working conditions The state of equipment laboratory safety and the adequacy of supplies are also assessed During OSE the technician observes the process for specimen collection smearing staining reading recording and reporting Stained smears will be reviewed during the visit When problems are detected solutions will be suggested and if possible implemented immediately It is the responsibility of the participating microscopy centers to assist the visiting supervisory staff The laboratory should make records available demonstrate routine performance and provide information The supervisor uses a standard checklist of items to be reviewed These visits provide an opportunity to learn about standards techniques and methods In addition they provide an opportunity for basic supervision including assessment of laboratory supplies basic procedures and performance of internal QC The supervisor will collect slides for rechecking deliver slides for panel testing or deliver results of EQA activities A format of OSE is given in Appendix 1 see
3. Slides Yes No Lens tissue Yes No Smearing staining equipment Yes No 0 1 Auramine Yes No 0 5 Acid alcohol Yes No 0 5 Potassium permanganate Yes No Slide boxes Yes No Study forms Yes No Other To be completed monthly during each study phase except during Validation phase every 2 week Module 9 Assuring Quality of Fluorescent Microscopy Page 14 of 15 IV Study procedures and documentation Item Adequate Problems identified Requirement Acceptable Study specific SOP followed Smear preparation observation Yes No Staining procedure observation Yes No Slides properly stained thickness Yes No field size Slides reading following grading Yes No chart Slide boxes stored Yes No All slides are available and stored as Yes No per lab register Storage of reagents reagent bottles Yes No labeled with content date of preparation date of expiry Storage of slides in boxes Yes No Study forms have correctly Yes No completed Completed study forms properly filed Yes No Review of 5 10 of results forms for Yes No correctness compared to source data Check 4 recent positive slides Check 20 IDs per visit V Internal Quality Control Control smears are used for each Yes No Comments new batch of staining solutions register are available Control positi
4. or 3 according to IUATLD WHO scale Number assigned by the supervisory site Module 8 Reading Recording and Reporting of Fluorescent Smears Page 9 of 12 Key messages PE Use the recommended grading scale for the FM smears Systematically scan the slide by moving across the smear in a horizontal direction Examine each field before moving on to the next field Read at least 30 high power fields 20x for FM smears before reporting a negative result Store all examined smears in the order they appear in the laboratory register Accuracy is critical at all levels of reporting and recording Each field must be filled out carefully Recording of results must adhere to guidelines provided in this module for FM smears The number of AFB found indicates how infectious the patient is so it is important to record exactly what you see Module 8 Reading Recording and Reporting of Fluorescent Smears Page 10 of 12 Review Module 8 Please answer the following questions based on the reading recording and reporting module How many AFBs are required for a 1 2 and 3 FM smears How many fields need to be examined when reading FM smears for AFB Which smears must be stored after examination When and how are microscope objectives cleaned Module 8 Reading Recording and Reporting of Fluorescent Smears Page 11 of 12 What are the 4 elements required for accurate recordkeeping What are the essential
5. Every 2 week Monthly Monthly Module 3 Use and Maintenance of the Primostar iLED Purpose Prerequisite Modules Learning Objectives Content Outline Handout and Exercises Appendices Module 3 Primo Star iLED To provide you with an understanding of the components and functionalities of Primo Star iLED its use and maintenance None At the end of this module you will be able to Name the essential components of a microscope and understand their function Correctly use brightfield and fluorescence applications of Primo Star iLED Maintain the instrument as per user manual Microscope components and operation Microscope maintenance Exercise Familiarization with Primo Star iLED by reading of several ZN and fluorescent slides Appendix 1 Specifications for a LED based fluorescence microscope Appendix 2 Questionnaire User Appraisal of Primo Star iLED Page 1 of 20 Module 3 Use and Maintenance of Primo Star iLED LED based fluorescence microscopy Replacing light microscopy with fluorescence microscopy would be one of the immediate options to improve the global TB situation A systematic review by WHO TDR and FIND has shown that a Fluorescence microscopy is on average 10 more sensitive than conventional light microscopy The increased sensitivity is greatest in low grade positives b The specificity is comparable c Reading a fluorochrome stained smear takes only
6. cost effective tool for laboratory diagnosis of patients with infectious TB smear positive pulmonary disease However if the laboratory diagnosis is unreliable then patients with infectious TB may not be diagnosed resulting in ongoing transmission of disease in the community and more severe disease in the individual Alternatively patients without TB may be treated unnecessarily Therefore quality assurance of AFB sputum smear microscopy is essential WHAT IS QUALITY ASSURANCE Accuracy and reliability of laboratory testing are critical to the success of TB control programs All parts of the testing system must be monitored to ensure the quality of the overall process to detect and reduce errors and to improve consistency between testing sites To ensure reliability and to reduce errors a quality system must address all parts of laboratory testing Quality Assurance QA is a system designed to improve the reliability and efficiency of laboratory services WHO and the International Union Against Tuberculosis and Lung Disease IUATLD have defined several components for a quality assurance program for AFB smear microscopy Quality Control QC A systematic internal monitoring of work practices technical procedures equipment and materials including quality of stains External Quality Assessment EQA A process to assess laboratory performance EQA includes onsite evaluation of laboratories panel testing and blinded smear rechecki
7. particles or streaks of mucous material process the sample but ensure that the poor quality of the sample is reported on the result form When possible encourage the patient to try again Even saliva can yield positive results All specimens should be processed except for broken or leaking containers which should be discarded safely and another specimen requested Module 4 Safety Precautions for TB Microscopy Page 10 of 13 Accept very small quantities if the patient has difficulty in producing sputum and if the aspect is right Blood streaked sputum is suitable but pure blood should not be examined Refer patients producing pure blood specimens immediately to a medical officer or doctor as they require emergency medical treatment Key message J g Good quality sputum samples are important for the diagnosis of pulmonary TB BA Early morning specimens provide the biggest yield of AFB For patients on treatment collect follow up specimens at intervals specified by the NTP Never collect sputum specimens in the laboratory Provide patients with clear instructions on the collection of good quality samples Patient referral specimen referral and slide referral are options for peripheral health centers not performing microscopy Assess the quality of all specimens submitted to the laboratory for microscopy Module 4 Safety Precautions for TB Microscopy Page 11 of 13 Review Module 4 Please answer the foll
8. recommended for diagnosis or by following NTP recommendations If the first two are positive then the third sample can be omitted For outpatients collect one sample at the time of presentation This is known as the spot specimen Give the suspect a second sputum container for collection the following morning and instruct the patient to deliver the morning specimen to the laboratory When the patient returns the morning specimen give him or her the third container and collect another spot specimen Give the patient clear instructions on the proper collection of a specimen for TB For hospitalized patients collect early morning specimens on three successive days since such samples often contain more bacilli and thus are more likely to be positive by microscopy Sputum collection for follow up of treatment For patients on treatment collect follow up specimens at intervals specified by the NTP Early morning sputum is the preferred specimen HOW TO COLLECT A SPECIMEN Sputum collection is the most dangerous procedure in the AFB smear microscopy laboratory and must be done in the open air and at a distance from other people Never collect sputum in the laboratory Give a new sputum container to each patient from whom sputum examination for TB is requested Demonstrate how to use it to collect a good specimen Clearly instruct the patient on the importance of sputum examination for diagnosis or follow up of TB how to open and cl
9. site once per month Au staining reagents by supervisory site Microscope for reading Conventional Brightfield 1000X Primo Star iLED 400X Conventional Brightfield 1000X Primo Star iLED 400X Primo Star iLED 400X Primo Star iLED 400X Microscope for re checking Conventional Brightfield 1000X Only for discrepants Primo Star iLED 400X Conventional Brightfield 1000X Conventional FM 200 250x where not available Brightfield after restaining 1000X Primo Star iLED 400X Primo Star iLED 400X Patient Frequency management of retrieving slides forms Based on ZN Once every result of 2 week microscopy center Based on Daily conventional FM result from supervisory site Brightlight if not available Daily provision of results Based on iLED Once every result from 2 week microscopy center Based on iLED Monthly result from microscopy center Supervisory visits with checklist Monthly Every 2 week Monthly Monthly Forms 1 Result Form ZN Baseline 2 Rechecking Form ZN Baseline 1 Proficiency Testing Result Form 2 User appraisal questionnaire 1 Result Form Validation 2 Rechecking Form Validation 1 Result Form Implementation 2 Rechecking Form Implementation Same as implementation Data transfer by courier At the end of phase Scanned by e mail following day
10. 25 of the time it takes to read a ZN stained smear To date the major constraints to the broader implementation of fluorescence microscopy are the high price for fluorescence microscopes and the lack of robustness and sustainability Conventional fluorescence microscopes use expensive and very fragile gas discharge lamps such as Xenon or Mercury lamps with high power consumption and a short lifespan of only 100 200 hours Furthermore the acceptability of darkrooms has generally been low The recent application of ultra bright LED light emitting diode technology to facilitate inexpensive fluorescence microscopy is a potentially significant advance in TB diagnostics for the following reasons e Low cost of ultra bright LEDs whose lifespan is 15 000 20 000 hours e Low power consumption plus possibility of battery operation Enhanced robustness e No need for air conditioning facility e No need for a dark room e Fluorescence stains do not require a heating step e Diagnostic performance 2 standard FM e Decreased technician workload The Primo Star iLED combines these advantages with high quality optics One of its major innovations compared to others is the use of ultrabright LED as a reflected light source permitting effortless switching from bright light to fluorescence light For complete microscope specifications refer to Appendix 1 MICROSCOPE COMPONENTS PRIMO STAR iLED wo w lt WWen
11. 30 g of phenol crystals in water e Mix both solutions by swirling for about one hour e Filter the Auramine solution by pouring through a funnel with filter paper held over the slides while transferring to a definitive container Working solutions have to be kept in dark bottles or better yet in a cupboard Label the bottle 0 1 Auramine add date and sign with initials The date first opened has to be mentioned Stocks and solutions should not be used over 1 month 1 liter of 0 5 hydrochloric acid in alcohol Decolorizing solution Hydrochloric acid technical grade 0 5 ml Ethanol 100 ml e Add 995 mL of 95 alcohol to a two liter Pyrex conical flask e Measure 5 mL of concentrated hydrochloric acid in a cylinder e Pour it slowly into the flask containing alcohol directing the flow of acid gently along the inner side of the flask with constant swirling e Mix well by swirling Label the bottle 0 5 acid alcohol add date and sign with initials The date first opened has to be mentioned Stocks and solutions should not be used over 6 months 1 liter of Quenching solution 0 5 permanganate e Weigh 5 g of potassium permanganate e Add the powder to 0 5 liter of distilled or purified water which has been placed in a conical flask e Swirl the contents of the flask to dissolve the dye e Add another 0 5 liter of water and mix again Label the bottle 0 5 Potassium permanganate add date and sign with initials The dat
12. 7 Please answer the following questions based on the smear preparation module What labelling information is needed on a slide What portion of the specimen should be used for smear preparation How can you determine the correct size and thickness of a sputum smear What are critical steps in the Auramine stain Module 7 Smear Preparation and FM Staining Methods Page 9 of 14 Laboratory Practical Session 3 Preparation of Smears Materials and Equipment Sputum specimens Glass slides clean with frosted end Disposable wooden or bamboo applicator sticks Spirit lamp burning spirit Bench disinfectant 5 phenol or 0 5 sodium hypochlorite Discard container Newsprint Forceps and Ruler marked in centimeters Procedure Review safety procedures in module 2 before beginning this procedure 9 10 11 12 13 14 15 16 USE a pencil to label the frosted end of a slide with the laboratory serial number on the sputum container OPEN the sputum container carefully and place the lid face up on the work surface EXAMINE the specimen to select the best portion to sample Choose yellow purulent or bloodstained particles if present PREPARE the smears near the flame of a spirit lamp The heat around the flame creates a sterile zone for aerosols generated during smearing of the specimen USE a wooden applicator stick to select the most purulent material from the specimen container USE the applicator
13. LZ Ea Controls of Primo Star iLED Fig 15 Legend oa wWDNDN O O ON 11 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Special eyecups with light protection Eyepieces Binocular body of the tube Tube Transmitted light reflected light changeover switch Brightfield Fluorescence Rotary knob for switching ON OFF and adjusting the illumination intensity for reflected light Carrying handle Plug in power unit IIlumination intensity indicators for transmitted light Rotary knob for switching ON OFF and adjusting the illumination intensity for transmitted light Fine focusing dial or knob right side Coarse focusing dial or knob right side Control knob for X travel of mechanical stage Control knob for Y travel of mechanical stage Clamping screw for condenser Transmitted light illuminator LED Slider with yellow filter with filter position for adapting the color temperature in transmitted light and with position for blocking the transmitted light path in case of reflected light fluorescence applications Luminous field diaphragm fixed Centering screws for condenser on condenser carrier Abbe condenser Fixed K6hler Objective Microscope stage Spring level of specimen holder Knurled ring of objective nosepiece Pilot lamp for reflected light fluorescence illuminator lighting blue when switched on brightness corresponds to intensity Lever for adjusting the apertu
14. NEVER collect sputum specimens in laboratories toilets waiting rooms reception rooms or any other enclosed space Always stand well clear and upwind when a patient is collecting a sputum sample LABORATORY Ideally the TB laboratory should be a well ventilated area which is dedicated to microbiology with restricted access Three separate areas are recommended for performing TB microscopy 1 Smear preparation and staining This area should be well lit and preferably near an open window to ensure adequate ventilation during smear preparation A sink with running water and a spirit lamp are also required An area of approximately six inches around the spirit lamp flame is considered as sterile zone as it coagulates any aerosol generated while opening of sputum containers and during smear preparation 2 Performing microscopy This area should have a flat bench or table for placing the microscope Subdued lighting is preferred If no electricity is available daylight must be used as the light source in this case place the microscope directly in front of a window 3 Record keeping and storage This third area is for entering data in the log book for Quality Control and for storing slides SAFETY PRACTICES IN THE TB MICROSCOPY LABORATORY Take the following precautions to protect yourself and all laboratory personnel Assume ALL specimens are potentially infectious Never smoke eat or drink in the lab Wash hands frequently
15. Primo Star iLED for TB diagnosis at microscopy centers without prior experience with fluorescence microscopy in low to moderate income settings and to identify barriers to implementation 2 To determine the false positivity and negativity rate of LED fluorescence reading compared to a ZN baseline and compared to results from the supervisory site 3 To determine the development of false positivity and negativity rates of LED fluorescence reading over time with increasing experience 4 To assess the impact of this implementation on daily workload and case detection rates for low middle and high volume settings 5 Determine lab technicians appraisal of using Primo Star iLED 6 To evaluate detailed costs associated with LED based fluorescence microscopy in comparison with conventional methods LED DEMONSTRATION PROJECT PHASES Study phase ZN Baseline Training Proficiency testing amp User appraisal Validation Proficiency testing amp User appraisal Implementation Proficiency testing amp User appraisal Continuation Duration 1 month 5 days 1 day Minimum 1 month Until targets met See above 3 months See above 6 months slides re checked 100 100 100 As per LQAS As per NTP Staining reagents Routine Zn stain For 10 Au and 10 ZN slides Au staining reagents provided by supervisory site once per month Au staining reagents provided by supervisory
16. Store in a cool and dry place Module 4 Safety Precautions for TB Microscopy Page 7 of 13 Key messages y g The greatest risk to a laboratory worker is a patient coughing a and not the patient s sputum specimen Never collect sputum in the laboratory Never smoke eat or drink in the lab Wash your hands frequently with soap and water at least before and after performing any procedures Gloves laboratory coats and surgical masks do not provide any appreciable protection against airborne transmission Protect the surrounding population by disposing of laboratory waste safely Avoid hazards that may occur in a TB laboratory by paying careful attention to safety procedures Always work carefully and in a safe manner Module 4 Safety Precautions for TB Microscopy Page 8 of 13 COLLECTION AND TRANSPORT OF TUBERCULOSIS SPECIMENS SUITABLE SPECIMEN CONTAINERS Use clean wide mouthed and leak proof specimen containers Single use disposable lastic containers 50 ml capacity are preferred THE NUMBER AND TIMING OF SPECIMEN COLLECTION To ensure optimal recovery of TB bacilli from sputum collect and process three specimens Consult your country s NTP for specific guidelines At least one should be an early morning specimen that can be collected by the patient upon rising Early morning specimens have the highest yield of AFB When TB is suspected collect three sputum specimens from the patient as
17. also the SOP Phase 2 Panel testing A panel test PT exercise usually involves sending a PT with an identical composition of negatives and positives to many laboratories at the same time It is useful as an initial gauge of the current level of laboratory performance as well as to determine critical priorities for expanding EQA While it also measures the ability of a technician to stain and or read smears it does not assess routine laboratory performance It is the responsibility of the laboratory technicians to read the PT slides in the same way they read patient slides They also need to take the same amount of time as for routine smears Panel testing is an opportunity to compare performance with other laboratories and reassures technicians that they can attain the same results as other laboratorians If discrepant results are found during PT this may require returning slides to the supervisory laboratory for rereading The proficiency testing during the project will be performed at the end of each study phase see Table 1 Slide re checking Re checking is the best method for evaluating performance and motivating staff to improve Re checking programs are intended to assess overall laboratory performance not to confirm any individual patient s diagnosis This process occurs when controllers at the Supervisory Site re read a 100 or a sample of routine smears from the microscopy centers During proficiency testing validation
18. and presented to WHO for policy recommendation on the use of these tests in high burden low income countries National TB Programs in countries participating in the demonstration projects for these new tests may of course independently make a policy decision on their use LED DEMONSTRATION PROJECT In collaboration with National TB programs and international organizations this demonstration project aims at a programmatic implementation and evaluation of the Primo Star iLED fluorescence microscope system in comparison to the existing microscope standard Participating microscopy centers will be grouped in clusters Each cluster will consist of one supervisory site and two to three microscopy centers The supervisory site will be responsible for training monitoring rechecking of slides and data management Hypothesis We postulate that the Primo Star iLED system is a feasible advantageous and cost effective replacement for ZN and where existing conventional fluorescence microscopy in low to moderate income laboratory settings Especially in busy microscopy centers it will increase the case detection rate while substantially decreasing the daily workload Endpoints The purpose of this demonstration project is to assess the implementation of Primo Star iLED as a ZN replacement for routine TB diagnosis in low and moderate income settings Specifically we are interested in the following 1 To assess the feasibility of implementing
19. compared to those of the available light microscope and where applicable fluorescence microscope Suggestions for improvements comments Question 6 Is the procedure for switching between brightfield and fluorescence light convenient and do you easily understand the symbols used for white light and fluorescence light LI Very convenient L Convenient L Not convenient Comments Sub question Do you consider the toggle switch to be robust enough C Yes L No Question 7 Is focusing with the fluorescence unit due to black background C Very difficult C Difficult but only a matter of training L Easy quickly got used to it Question 8 Do you use the option of opening the slider on the white light source to focus with the fluorescence unit dark background gets structured which makes focusing easier L Yes always use this to facilitate focusing L Sometimes LC Never Question 9 Are the blue LEDs on both sides of the microscope that indicate the intensity level of the brightfield illumination convenient or rather disturbing dazzling L Convenient L Disturbing Dazzling Question 10 Are the 4pcs objectives with magnifications 10x 20x 40x and 100x the best choice for the applications Auramine O fluorescence and Ziehl Neelsen brightfield detection of pulmonary tuberculosis LI Yes L No would prefer to have a magnification Comments Question 11 Which magnification do you prefer for fluor
20. data required in the Laboratory Register Why is it important to identify whether a specimen is for diagnosis or follow up What are the quantifying categories of smear reading What are the consequences of reporting a false positive result Module 8 Reading Recording and Reporting of Fluorescent Smears Page 12 of 12 Module 9 Assuring Quality of Fluorescence based AFB Microscopy Purpose Prerequisite Modules Learning Objectives Content Outline Handout and Exercises Appendix To provide you with an understanding of quality assurance and external quality assurance of AFB smear microscopy Modules 1 8 At the end of this module you will be able to Describe the elements of Quality Assurance Explain why internal Quality Control is important in assessing laboratory performance Describe the three components of External Quality Assessment EQA Prepare for a supervisory visit Describe the process of sampling slides for blinded smear rechecking What is Quality assurance Quality Control in the TB laboratory EQA and why it is important EQA components e Panel Testing e On Site Evaluation e Blinded Rechecking Exercise 1 2 and 3 Appendix 1 Onsite Evaluation Checklist Module 9 Assuring Quality of Fluorescent Microscopy Page 1 of 15 Module 9 Assuring Quality of Fluorescence based AFB Microscopy For many countries with a high burden of TB direct smear microscopy remains the most
21. indicator LEDs to indicate the mode of operation The right green LED indicates the availability of the power supply line The left yellow LED indicates the charging mode of the accumulators When the yellow LED is on the battery pack is charging When it is off charging is completed The battery pack switches off automatically when the light sources of the microscope are turned off The battery pack turns off automatically when a critically low charging level is reached In this case the battery pack needs to be connected to the power supply to charge The battery pack will require charging after 6 8 hours of use OPERATION OF PRIMO STAR iLED for more details see User Manual The correct operation of the microscope is as important for the quality of results as applying correct smear and staining procedures Switch on the light 5 at low intensity the level of light intensity is indicated by small blue LEDs 6 to the right of the switch Place a specimen slide on the stage Be sure the slide is not placed upside down The next steps depend on whether you wish to use the fluorescent or brightfield mode Fluorescent mode When the lever on the fluorescence unit 8 is turned to the front of the microscope the fluorescence mode is active You can adjust light intensity using the knob 9 behind the lever The transmitted light slider 10 must remain closed in the fluorescence mode otherwise the contrast of fluorescenc
22. items prior to disposal Fresh household bleach diluted 1 10 with water approximately 5 sodium hypochlorite can also be used as a general disinfectant Bleach solution works well for cleaning up blood spills however it is somewhat less effective than phenolic agents against TB It is important that a bleach dilution be made fresh since it loses potency with time Seventy percent alcohol is a good agent for cleaning bench tops Module 4 Safety Precautions for TB Microscopy Page 4 of 13 Surgical masks do NOT protect against TB infection as TB bacilli can pass through these masks Therefore surgical masks provide a false sense of protection Effective respiratory protection such as an N95 respirator is expensive and unnecessary if the technician uses appropriate technique Gloves are not required for use in smear preparation since TB infection is acquired by airborne inhalation Each country must evaluate the risks and decide on the level of protection that is appropriate with the resources that are available Hand washing and careful techniques are mandatory for safe laboratory practice in all countries Take the following safety precautions before and during laboratory procedures Reject broken or leaking containers Request another specimen Once collected allow a sputum specimen to stand undisturbed for at least 20 minutes before opening to allow any aerosols to settle Cover sputum containers with the
23. ordered items arrive at the laboratory check to ensure that what you ordered was delivered and that items are in good condition i e expiry dates Label each item with the date you received it when you open and use it be sure to mark those dates on the item also Place items on shelves whenever possible Items should be stored in an orderly fashion keeping like items together Store new shipments behind existing shipments and make sure that the oldest items are stored in front so they will be used first Store all stock items in a well ventilated clean and tidy room Store the chemicals staining reagents and other reagents away from direct sunlight Lastly be sure to update the stock management records Keep staining reagents in well closed bottles out of direct sunlight and preferably inside a cabinet unless they are made of dark glass or plastic Label all bottles containing staining reagents with name and date of preparation Well prepared staining solutions have a shelf life of 3 6 months if they are kept in the dark Old reagents deteriorate and may not work effectively For demonstration project purposes Auramine staining solution will be kept for lt 1 month Acid Alcohol and Potassium Permanganate solutions for lt 3 months When storing new microscope slides make sure they are as dry as possible to prevent fungus growth Keep new sputum containers in closed cartons or bags Microscopes should be kept in a well ventilated dry
24. satisfied are you with the color impression for ZN stain of the Primo Star iLED white LED in comparison to a standard light microscope halogen bulb L AFBs can be better distinguished C Same L AFBs can be less well distinguished Comments Question 3 How satisfied are you with the resolution and depth of focus of Primo Star iLED C Very satisfied better than for the available light microscope and where applicable fluorescence microscope L Satisfied comparable to available light microscope and where applicable fluorescence microscope CI Not satisfied not as good compared to those of the available light microscope and where applicable fluorescence microscope Question 4 Was there a difference between the homogeneity of fluorescence illumination in the field of view compared to your standard microscope L Field of view of Primo Star iLED is more homogenously illuminated L Same L Field of view of Primo Star iLED is less homogenously illuminated Question 5 How satisfied are you with the overall handling features of the microscope on off switch intensity regulation of bright light and fluorescence light variable viewing height focus mechanism coarse and fine focus L Very satisfied better than for the available light microscope and where applicable fluorescence microscope L Satisfied comparable to available light microscope and where applicable fluorescence microscope C Not satisfied less good
25. shape from very short fragments to elongated types They may be uniformly stained or with one or many gaps or even granular They occur singly or in small groups and rarely in large clumps The typical appearance is of bacilli that are rather long and slender slightly curved rods With good staining always check first a freshly stained positive control there may still be fluorescing sometimes green artifacts which do not have the typical shape Also non fluorescing bacillary shapes must be considered as artifacts Recording of Results Semiquantitative results will be recorded according to the NTP guidelines If no local guidelines are available it is recommended to follow the below grading scale Module 8 Reading Recording and Reporting of Fluorescent Smears Page 3 of 12 Table 1 Recommended number of AFBs and fields for grading of ZN and fluorescent stained Slides IUATLD WHO MICROSCOPY SYSTEM USED SCALE 01000x BRIGHTFIELD FLUORESCENCE FLUORESCENCE field HPF 1000x magnification 1 200 250x 400x magnification 1 length 2cm 100 magnification 1 length length 40 fields Result HPF 30 fields 300 HPF 200 HPF Negative Zero AFB 1 length Zero AFB 1 length Zero AFB 1 length Scanty actual count 1 9 AFB 1 length or 100 HPF 10 99 AFB 1 length or 1 29 AFB 1 length 1 19 AFB 1 length 1 100 HPF 1 9 AFB 10 30 299 AFB 1 length 20 199 AFB 1 length fields 24 1 10 AFB 1 HPF on 10 100 AFB 1 field 5 50 AFB 1
26. staining working solutions required for Auramine staining from their respective supervisory site on a monthly or quarterly basis The supervisory sites will be responsible for timely ordering of all ingredients required to prepare the staining solutions for the 2 3 participating microscopy centers as well as for their own needs The supply mechanism for general supplies such as sputum containers slides etc will not change and will be handled as per NTP guidelines SUPPLY LIST FOR FLUORESCENCE BASED AFB MICROSCOPY Auramine stain The following is a list of general supplies required at AFB microscopy centres using Auramine staining method Items Quantity Sputum containers and slides 1 per examination Burning spirit for heat fixation of smear only 0 5 ml per smear Marker pens or grease pencils to label 1 per 2 months sputum containers smears Tissue paper to clean microscope lens 20 sheets per month lens paper or soft toilet paper 1 roll per month Forceps 1 per 5 years Staining rack 1 per 5 years Drying rack 1 per 5 years Safety glasses if handling concentrated 1 per 5 years acids Spirit lamp or Bunsen burner 1 per 10 years Slide boxes 100 slides Need to have enough to store all smears collected during 12 months Spare bulb for Primo Star iLED 1 per 10 years Disinfectant to clean bench top 100 ml per month Module 5 Managing Supplies Page 2 of 11 General supplies requiring NTP specification Certain supply items requ
27. stick to transfer the selected specimen particles fluid to the glass slide SMEAR the specimen over a 2 x 3 cm area in the middle of the unfrosted part of the slide USE the applicator stick to crush break up and spread out particles USE small circular motions to distribute the specimen evenly DISCARD the applicator stick into a waste receptacle containing a suitable disinfectant RESEAL the sputum container and set aside ALLOW the smear to air dry completely never use heat to reduce smear drying time AFTER the slide is completely dry hold the slide using forceps with the smeared slide facing upwards Pass the slide over the flame 2 3 times about 2 3 sec each time EVALUATE the fixed smear for the proper thickness Consider smear to be potentially infectious until after it has been stained Make sure you WASH your hands before leaving the laboratory Note Use these smears for Practical Session 4 Staining Sputum Smears Module 7 Smear Preparation and FM Staining Methods Page 10 of 14 Laboratory Practical Session 4 Staining Sputum Smears Materials and Equipment Ten smears prepared in Practical Session 3 and a set of 5 unstained panel smears Staining sink Running water Small funnel with filter paper Set of Auramine stain reagents for each staining area Spirit lamp Beaker forceps and gloves Procedure 10 ARRANGE the slides by placing them in serial order on the leveled staining bridge smea
28. the specimen slide by turning only the fine focus adjustment knob Use only the 100x objective immersion objective for observation through immersion oil All other objectives must be used without immersion oil and kept dry Read at least 100 high power fields before reporting a negative result Note Fewer than 100 fields may be read if the slide is found positive for AFB Usually examining 100 fields takes about 5 minutes To view the next slide the entire procedure does not need to be repeated Turn away the 100x objective and take out the slide add a drop of immersion oil on a new stained smear and insert onto stage then turn to 100x objective Applying immersion oil when using the brightfield option Make sure that the smear is facing upwards when the slide is placed on the mechanical stage Put one drop of immersion oil on the stained smear letting it fall freely onto the slide Never allow the oil applicator to touch the slide Touching the slide with the applicator could lead to contamination of the oil with AFB and could transfer AFB to a negative slide MICROSCOPE MAINTENANCE Never attempt to disassemble any part of the microscope for repair If there is any problem with the microscope contact the microscope company s technical support unit or a qualified technician Treat the microscope with care Never expose it to sharp knocks vibrations moisture dust or direct sunlight Humidity causes fungal growth on the s
29. After device designed to attach to a bright field microscope are or will become available shortly One of the major innovations of Primo Star i LED compared to others is the use of ultrabright LED as a reflected light source The new microscope has high quality optics and is very robust e g complete antifungal coating It allows effortless switching from bright light to fluorescence light and can be battery operated These innovations in combination with the affordable price may allow wide introduction of fluorescence microscopy and gradual replacement of conventional microscopy in the public health sector of resource limited countries In collaboration with National TB Programs and International Partner Organizations the Foundation for Innovative New Diagnostics FIND is undertaking this large scale demonstration project to explore the feasibility and impact of scaling up use of LED fluorescence microscopes to improve TB control These training modules have been developed for the FIND LED demonstration project and are based on the Acid fast Direct Smear Microscopy Training Modules developed by WHO CDC RIT IUATLD APHL in 2004 In addition the training modules developed by Fujiki A 16 and the Central TB Division DGHS MoHFW Government of India New Delhi 17 helped in developing module six References 1 Mark D Perkins Giorgio Roscigno Alimuddin Zumla Progress towards improved tuberculosis diagnostics for developing countr
30. FIND foundation for innovative new diagnostics TRAINING MANUAL FOR FLUORESCENCE BASED AFB MICROSCOPY Demonstration Project iLED Effectiveness of the Primo Star iLED Microscope for Detection of Tuberculosis Technical and Financial Agency Foundation for Innovative New Diagnostics Study Coordinator Dr Catharina Boehme email catharina boehme finddiagnostics org Project leader Dr CN Paramasivan email cn paramasivan finddiagnostics org Partnering for better diagnosis for all Based on Acid fast Direct Smear Microscopy Training Introduction Module developed by WHO CDC RIT IUATLD APHL in 2004 Demonstration Project iLED Training Manual Version 1 0 1 September 2008 Other contributors include Dr Pamela Nabeta Dr VH Balasanghameshwara Confidentiality statement The information contained herein is the property of FIND and may not be reproduced published or disclosed to others without written authorization Module Contents Introduction The Possible Role of LED based Fluorescence Microscopy in Improving the Global Tuberculosis Situation Demonstration Project Primo Star iLED Study Outline Use and maintenance of the Primo Star iLED Safety Precautions for TB Microscopy Including Collection and Transport of Sputum Samples from TB suspects Managing Supplies for Fluorescence based AFB Microscopy Preparation of Reagents for Fluorescence based AFB Microscopy Smear Preparati
31. O per slide 0 003 liter See below Acid Alcohol per slide 0 006 liter 107017 Ethanol absolute 2 5 Permanganate per slide 0 003 liter 109057 Hydrochloric acid 1 Staining solution requirements month I quarter 105080 Potassium permanganate 1 kg 0 1 Auramine O 0 O liter 0 5 Hydrochloric Acid Alcohol 0 O liter 0 5 Permanganate 0 O liter Required Actual required Actual aa ai P Banter Sa or NER stock oi han Product units order Unit quarter subtracted round up Auramine O powder g 1 0 1 bottle 50 g 0 bottles Phenol crystals g 30 0 1 bottle 500 g 0 bottles Ethanol for Auramine O I 0 1 0 Ethanol for Hydrochloric Acid I 0 995 0 Ethanol combined I 0 0 1 bottle 2 51 0 bottles Hydrochloric Acid I 0 005 0 0 1 bottle 11 0 bottles Potassium permanganate powder g 5 0 i 0 1 bottle 1000 g 0 bottles Estimated general supplies for next Required poet STN Actal A Per smear for next stock on hand Product Units Order Unit quarter including 20 reserve quarter subtracted round up Sputum container 1 0 0 1 bag 1000 0 bags Slides 1 0 0 1 box 72 0 boxes Slide Boxes 1 0 0 1 box 1 0 boxes Burning Spirit for heat fixation l 0 0005 0 0 1 bottle 11 0 bottles MODULE 5 STOCK BOOK Item Name Unit AIVEUE Date count eM Date of Quantity Date Total stock count units performed reques
32. agm Open the diaphragm properly is closed The light cannot be switched on Contrast Problem Solution FL You are in brightfield mode Switch to fluorescence mode by turning the lever towards fluorescence BF You are in fluorescence Switch to brightfield mode by turning the lever mode towards bright light BF FL The microscope has no Plug in the cable or connect and switch on the power supply battery pack BF FL The LED bulb is defect Replace LED by following instructions in user manual There are dark shadows in the field which move as you turn around the eyepiece Problem Solution Surface of the eyepiece has scratches Replace the eyepiece Eyepiece is dirty Clean the eyepiece The image with the high power objective is not clear Problem Solution Slide is upside down Turn the slide over There is dirt on the objective Clean the lens There is an air bubble in the oil Move 100x lens quickly from side to side Oil is too sticky Use thinner or specified immersion oil The image with the low power objective is not clear Problem Solution There is a layer of dust on the upper surface of the objective Clean the lens There is oil on the lens Clean the lens If the view field is still dim and cloudy consider the following possible causes Massive growth of fungus on the lenses or pr
33. amine O g Phenol g Alcohol ml Deionized H20 ml Decolorizing solution Hydrochloric acid Hydrochloric acid ml Alcohol ml Quenching solution Potassium permanganate g Deionized H20 ml Module 6 Preparation of FM Reagents Page 8 of 11 Appendix 2 Example of Logbook for Quality Control of Auramine O Staining Reagents Batches checked on date 3 5 08 Auramine O Au O batch 3 5 08 Hydrochloric acid batch 3 5 08 Potassium permanganate batch 3 5 08 Average grading positive controls LED 40 x objective no 345 90 40 fields no 411 66 40 fields Control slide AFB color AFB number Decolorization Decision 345 13 Bright yellow 80 40 40x F OK Accept Au O 411 26 Bright yellow 60 40 40x F OK Accept Au O NEG NA None in 30 40x F OK Accept others NEG NA None in 30 40x F OK Accept others Note Batches checked on date 17 5 08 Au O batch 17 5 08 Hydrochloric acid batch 17 5 08 Potassium permanganate batch 17 5 08 Average grading positive controls LED 40 x objective no 345 90 40 fields no 411 66 40 fields Control Slide AFB color AFB number Decolorization Decision 345 16 weak yellow 2 30 40x F OK Reject Au O 411 29 NA 0 30 40x F OK Reject Au O NEG NA None in 40 40x F OK Accept others NEG NA None in 40 40x F OK Accept others
34. and safe place Optical parts must be kept in a dry place to prevent damage from fungus STOCK MANAGEMENT Stock management means properly maintaining adequate supplies to ensure uninterrupted service It involves performing a stock count physical inventory maintaining proper inventory records determining how much to order when to order placing orders properly inspecting and verifying supplies received and ensuring proper storage of stock Stock management ensures the availability of staining reagents and materials avoids the use of old reagents and minimizes waste The availability of high quality microscopy testing services depends on the uninterrupted availability of supplies required for testing It is important not to under or over stock supplies Under stocking will result in insufficient supplies and will interrupt the testing process Over stocking presents different problems Laboratories have limited space that excessive stock can overwhelm compromising safety and security Excessive stock also requires additional management which takes up laboratorians valuable time Over stocking can lead to deteriorated reagents and waste For these reasons proper management of your stocks is very important and will ensure that only adequate supplies are on hand Module 5 Managing Supplies Page 4 of 11 RECORDKEEPING STOCK SUMMARY FORM AND STOCK BOOK An inventory count is performed to know exactly what and how much stock is on han
35. and validation phase 100 slides will be re checked A minimum sample of slides will be rechecked as per LQAS method during implementation phase The sample will be based on 80 sensitivity 100 specificity and 0 acceptance number for a pre selected ranges of slide positivity rate and annualized negative slide volumes see Table 2 below Module 9 Assuring Quality of Fluorescent Microscopy Page 5 of 15 Table 2 Lot quality assurance sampling LQAS for implementation phase 80 sensitivity 100 Specificity and 0 Acceptance number Slide positivity rate SPR Annualized no of negative slides ANSV at ie 2 5 4 9 5 0 7 49 7 5 9 9 10 14 9 gt 15 demonstration site Monthly sample size of randomly selected slides to be re checked 301 500 22 14 12 10 8 501 1000 28 18 12 10 8 gt 1000 40 20 14 10 8 Slide positivity rate and annualized negative slide volumes are calculated based on the data available for one month from each of the microscopy centers during ZN baseline phase The sample size for each microscopy center is selected based on SPR and ANSV as per Table 2 given above Re checking Re staining will only be required for Auramine O fluorescent stained smears if staining is considered of low quality by the supervisor Feedback on the results of discordant slides along with the slides must be returned to the supervisor during monitoring visits and action taken to resolve any performance problems ident
36. chnician is at considerably more risk when sputum is processed for culture and drug susceptibility testing These procedures require shaking and centrifugation Consequently special equipment such as biological safety cabinets and Biosafe centrifuges which are costly to purchase and maintain are required However this equipment is not justified for the AFB smear microscopy laboratory Transmission of TB bacilli The TB bacilli are almost always transmitted by patients with active pulmonary disease The patient expels TB bacilli in small droplets of respiratory secretions These secretions quickly evaporate leaving droplet nuclei of less than 5 um in diameter Droplet nuclei of this size containing 1 3 bacilli can remain suspended for long periods of time in the air and following inhalation are able to reach deep into the lungs to produce infection Larger particles do not remain airborne for as long and do not transmit tuberculosis as efficiently The risk of infection depends on 1 the infectiousness of the source 2 the environment e g overcrowding and inadequate ventilation promote transmission of droplet nuclei 3 the duration and intensity of exposure and 4 the susceptibility of the recipient Smear positive patients have 10 10 bacilli per millilitre of sputum whereas smear negative patients have about 10 or less per millilitre This difference in bacterial load as determined by smear status and radiologic exte
37. ck book Inspect and verify supplies received Explain storage of supplies Supply list for smear microscopy Supply storage Stock management Recordkeeping Stock book use and importance Calculating supplies required Placing receiving and storing supply orders Stock Management Spreadsheet Stock Book Stock Summary form Exercise 1 Calculation of quarterly supply requirements for a supervisory centre that prepares staining reagents for itself and 3 microscopy sites Not mandatory Exercise 2 Calculation of quarterly supply requirements for a microscopy centre that receives prepared staining reagents None Module 5 Managing Supplies Page 1 of 11 Module 5 Managing Supplies for Fluorescence Microscopy LABORATORY SUPPLY SYSTEMS Laboratory supply systems vary among countries Factors that affect how an AFB microscopy laboratory receives its supplies include whether the health care system is integrated or vertical whether the laboratory calculates its own needs and places its own orders or whether the laboratory receives orders based on calculations performed at another level in the health care system In any case microscopy laboratories must know how to perform orders how to ensure that required supplies are always available for testing and how to store such supplies SUPPLY SYSTEM DURING LED DEMONSTRATION PROJECT For the duration of the LED demonstration project participating microscopy laboratories will receive the
38. continuation phase List the microscopes used for each of the phases ZN baseline validation implementation and continuation phases List the frequency of OSE during each of the phases ZN baseline validation implementation and continuation phases Module 9 Assuring Quality of Fluorescent Microscopy Page 12 of 15 In which phase the decision to start the patients on treatment is based on results of supervisory site List the frequency and number of slides for Proficiency Panel testing for each of the phases Module 9 Assuring Quality of Fluorescent Microscopy Page 13 of 15 e foundation for innovative new diagnostics Appendix 1 On Site Evaluation Checklist ON SITE EVALUATION CHECKLIST SUPERVISORY VISIT User Supervisory Site iLED Demonstration Project l General Information Supervisor Supervisory Site name ID Microscopy Center name ID Date of Visit ll Laboratory infrastructure and equipment Uninterrupted power supply No problems Rare and short interruptions _ Regular or sometimes long interruptions _ Running water supply No problems Rare and brief interruptions _ Regular or sometimes long interruptions _ Primo Star iLED microscope No technical problems observed _ Technical problems observed _ Specify ll Adequate stock within expiry dates and supply of Item Adequate Comments Requirement
39. d It means the physical counting of each item in the stock and it should be performed at the end of each quarter A designated person is responsible for performing this count The quantities of items on hand can be recorded on a form such the Stock Summary form found at the end of this module This form is also useful to record the work performed number of smears examined by a microscopy centre Determine the work performed at the end of each quarter This can be done by reviewing the TB register Efficient stock management depends upon accurate recordkeeping Keeping accurate records ultimately saves time Proper inventory records help laboratorians determine and predict their pattern of consumption and estimate supplies for a year for budgeting purposes The stock book contains a list of all items in the store It must be routinely updated when orders are placed and received It also serves as a reference to track orders that have been placed and not received The information recorded in the stock book regarding when orders are placed and when they arrive may help a site to adjust reserve supplies that are kept on site to ensure uninterrupted testing CALCULATING SUPPLIES REQUIRED Calculations for the supplies required for a microscopy centre can be based on the actual number of smears examined during a month and a stock count of supplies on hand This actual supply calculation is typically performed with a spreadsheet For the LED dem
40. d the patient is available ask the patient for the required information Also the NTP needs to know whether the specimens are for diagnosis or follow up Module 8 Reading Recording and Reporting of Fluorescent Smears Page 5 of 12 A completed Laboratory Request Form should give the following information See Appendix 3 Name of health center Date Patient s name address age and sex Source of specimen Reason for exam diagnosis or follow up Specimen ID number Signature of person requesting exam Microscopy report After the sputum smear has been read the result should be written immediately into the result form Whenever possible use a RED pen for positive results Check that the number on the slide matches the number on the Laboratory Request form Subsequently the results are written onto the Laboratory Register again checking to make sure that the laboratory serial number matches for both The microscopy report should include the following information Specimen ID number laboratory serial number Date of specimen collection Evaluation of the quality of the specimen e g bloody mucopurulent saliva Smear result Date of examination Name and Signature of technician who performed the microscopy Once completed the microscopy report should be made available as soon as possible preferably no longer than 24 hours after the laboratory receives the specimen The Laboratory Register It is rec
41. d the smear about 4 5 cm over a piece of printed paper If letters cannot be read it is too thick Figure 5 3 Air drying of smear Allow the smear to air dry completely at room temperature Figure 4 Do not dry smears in direct sunlight or over a flame 4 Heat fix smear After the slide is completely dry use forceps to hold the slide upwards Pass the slide over the flame 2 3 times for about 2 3 seconds each time Figure 3 Do not heat or keep the slide stationary over the flame for too long or else it will be scorched A well stained smear should have more than 20 leucocytes of uniform size of 2 x 3 cm with even good thickness and should be properly decolorized Graphic representations of smear preparation are shown below Figure 1 Module 7 Smear Preparation and FM Staining Methods Page 3 of 14 Figure 2 Figure 3 Module 7 Smear Preparation and FM Staining Methods Page 4 of 14 Figure 4 Japenese pair In i lim d Be ina duada al Figure 5 Module 7 Smear Preparation and FM Staining Methods Page 5 of 14 STAINING WITH AURAMINE O SOLUTION Principle of acid fastness The nucleic acids of the cell become stained by Auramine O which later does not lose its color despite the action of acids or alcohol Auramine O staining procedure 1 AON NON 9 10 Arrange slides in serial order on staining bridge with smear side up at a distance of at least 1cm between every slide F
42. damage or break slides e Condenser Abbe type condenser 0 9 1 25 with iris diaphragm e Objective 10x 20x 40x 100x oil immersion colour corrected infinity optics e Eyepieces wide field 10x 18 mm FOV 18mm adjustable can be used by spectacle wearers e Brightfield illumination in transmitted light mode White light LED minimum 3W e Fluorescence illumination in reflected light mode Blue light LED minimum 3W e Focus Focus drive must be a self tensioning three ball design Coarse and fine focusing dials or knobs on both sides e Power supply wide range input 100 240V 50 60 Hz e All gears throughout the microscope mechanical stage focus condenser rack and pinion must be made of metal brass stainless steel or aluminum no plastic components e Ergonomic design e Anti fungus treatment e Microscope has to fulfil the following standards CE CSA UL IvD ISO 9001 Appendix 2 Questionnaire Appraisal of Primo Star iLED Trial Site Name where applicable Supervisory Site Country Date of completion DD MM YY Completed by First name last name Position Microbiologist laboratory technologist microscopist Instructions This questionnaire should be completed by at least 2 staff members per supervisory site and 1 from each microscopy center at the end of each demonstration project phase Please check for each question the box of your selected evaluation category Please provide further details in t
43. ds splashes of acid causing burns to the skin or eyes Do not use alcohols near an open flame as they are flammable Phenol is a toxic chemical Avoid direct contact with the skin or mucus membranes Reduce exposure to phenolic fumes by staining smears in a well ventilated area and by limiting the number of slides in each staining batch to a maximum of 12 Module 4 Safety Precautions for TB Microscopy Page 6 of 13 HANDLING AND STORAGE Auramine O Storage Tightly closed in a well ventilated place Storage temperature 5 C to 30 C Auramine prepared stain should be stored in amber coloured bottles for a maximum period of one month for the study purposes Concentrated Hydrochloric acid Handling Wash thoroughly after handling Remove contaminated clothing and wash before reuse Use with adequate ventilation Avoid contact with skin or eyes Do not ingest or inhale Storage Keep away from heat and flame Do not store in direct sunlight Store in a cool dry well ventilated area and away from incompatible substances Phenol Phenol should be stored in a cool dry well ventilated area in tightly sealed containers Containers of phenol should be protected from physical damage and ignition sources and should be stored separately from strong oxidizers especially calcium hypochlorite acids and halogens Potassium permanganate Keep tightly closed Keep away from combustible materials heat sparks and open flame
44. e first opened has to be mentioned Stocks and solutions should not be used over 6 months Module 6 Preparation of FM Reagents Page 3 of 11 Next steps Let the flasks with freshly prepared reagents stand covered until quality control procedures have been performed After these reagents have passed quality control pour the solutions into clean bottles and label them If bottles are reused clean thoroughly use acid alcohol and a bottlebrush to remove this residue On the label of the bottle clearly print the reagent name concentration and the preparation date STORAGE OF REAGENTS Well prepared reagents will keep for at least six months to one year even at higher temperatures However for demonstration project purposes Auramine staining solution will be kept for lt 1 month and acid alcohol and potassium permanganate solutions for lt 3 months Store all reagents in clean and tightly closed bottles with a label showing the name of reagent and the date of preparation Keep these bottles out of direct sunlight If clear bottles are used keep stocks of reagents in a closed cabinet QUALITY CONTROL Preparation of positive and negative controls Quality control QC smears which are supplied by FIND are manufactured validated and unstained slides After the demonstration is completed the NRL of your country will provide these slides In case you need to prepare your own QC slides make positive control smears with low posi
45. e is poor The slider may be opened initially to increase the background signal and thereby facilitate focusing By loosening the retaining ring the slider can be turned to the desired direction for a better handling Focus the specimen with the 10x or 20x objective by turning the coarse adjustment knob 7 Adjust the distance between the ocular lenses until both the right and left images become one Fine focus the image by turning the fine adjustment knob 7 Change to the 40x objective for screening the slide Focus the specimen slide if necessary by turning only the fine focus adjustment knob 7 Scan the smear by moving across the smear in a horizontal direction Stop and observe each field before moving onto the next field Read at least 40 high power fields before reporting a negative result Note Fewer than 40 fields may be read if the slide is found positive for AFB Brightfield mode 13 14 18 19 20 21 By turning the lever 8 clockwise to face the rear the microscope switches automatically to brightfield illumination mode The intensity of the brightfield illumination can be adjusted using the fine adjustment knob 5 Focus as in fluorescent mode steps 6 9 Put one drop of immersion oil on the smear Change to the 100x objective Be sure the condenser is raised as high as possible to maintain the intensity of the light Open the condenser iris to 70 80 of the aperture diameter Focus
46. e stained slide to dry in direct sunlight When the slides are completely dry they are ready for microscopy If they are not read immediately place them in a slide box Module 7 Smear Preparation and FM Staining Methods Page 11 of 14 Module 8 Reading Recording and Reporting of Fluorescent Smears Purpose Prerequisite Modules Learning Objectives Content Outline Handout and Exercises Appendices To provide you with an understanding of smear reading recording and reporting of fluorescent smears Module 3 and 7 At the end of this module you will be able to Describe the method for observing AFB using 40x objective for Auramine O stained smears Recognize the appearance of AFB in a stained smear Describe the quantification scheme for reporting results Appropriately quantify results in the study forms Required materials Reading the smear Recording of results WHO IUATLD grading scale Storing smears Essential elements of record keeping Reporting results Exercise Laboratory Practical session 5 Reading and reporting of panel slides from Laboratory Practical session 4 Appendix 1 Results form Module 8 Reading Recording and Reporting of Fluorescent Smears Page 1 of 12 Module 8 Reading Recording and Reporting of Fluorescent Smears Examination of sputum smears for acid fast bacilli requires a good microscope and a motivated trained technician Required Materials The following mater
47. e treatment may not be extended resulting in inadequate treatment and potential drug resistance False positive consequences False positive means that the result reported as positive was actually negative Patients are treated unnecessarily Treatment may be continued longer than necessary Medications will be wasted Storing Smears Store ALL slides in slide boxes in the order they were recorded in the laboratory register This will allow easy sampling of slides for external quality assessment using blinded slide rechecking Do not write the result on the slide Module 8 Reading Recording and Reporting of Fluorescent Smears Page 7 of 12 Exercise Laboratory Practical session 5 Reading and reporting of panel slides from Laboratory Practical session 4 Read and report the smears prepared in Practical Session 4 Use Appendix 1 Microscopy recording form Appendix 2 Grading chart and Appendix 3 Panel testing report form Module 8 Reading Recording and Reporting of Fluorescent Smears Page 8 of 12 Appendix 1 e foundation for innovative new diagnostics Results Form For Laboratory Practical Session 5 Microscopy Center ID Supervisory Site ID Slide ID Date of sample Date of slide reception reading Results Neg Pos Lab Tech ID If negative tick neg if positive register scanty 1 2
48. ed in another laboratory indicate date received Record any material found to be unsatisfactory and remove it from the laboratory immediately so it is not used Limit stocks to three months supply For study purposes Auramine solution supply will be limited to one month Rotate stock to ensure that oldest material is used first Staining and smear examination FM staining In addition to the above mentioned activities the following should be observed Do not heat Auramine O solution Do not use oil for reading Do not keep stained slides in direct sunlight Unacceptable control slides mean e positive control is not stained bright yellow e negative control remains fluorescent even after decolorization e background is not dark Resolve any problems with control smears before reporting patient smears Some problems may require repeating patient smears in a failed staining batch Module 9 Assuring Quality of Fluorescent Microscopy Page 3 of 15 Recording and Reporting Send microscopy results out as soon as they are available preferably within 24 hours after the sputum specimen is received Monitor any delays or turn around time in delivery on the report form Analyze microscopy results on a monthly basis to detect changes which may indicate a problem All microscopy results must be recorded in standard format in laboratory register It is recommended that all records be retained for at least two years For study specific form
49. erculosis control program Records should include information about the following events e What type of specimens were received by the laboratory e How were the specimens identified e How results are reported e When specimens are sent to higher level laboratories for culture and drug susceptibility testing Laboratories should use a standardized record keeping system that is simple practical and limited to recording only essential information Accurate recordkeeping is based on four fundamentals e Completeness e Consistency e Credibility e Timeliness During the project supervisors should review 5 10 of the study results forms for correctness compared to source data Laboratory Register and ensure that laboratory recordkeeping meets the above elements Record results in the laboratory register and study forms immediately after reading smears LABORATORY REQUEST AND REPORT FORMS Patient details In many countries the Laboratory Request Form and the Microscopy Report Form are combined into a single sheet of paper This enables better tracking of reporting and not only reduces the time it takes to transcribe patient and sample related information on separate report forms but also reduces transcription errors A Laboratory Request Form must be submitted with the first sputum specimen or patient Information on the form must exactly match the information on the slide of the specimen container If the form is incomplete an
50. escence detection of AFBs 20 times or 40 times L 20x C 40x Question 12 In your opinion can Primo Star iLED be used without a darkroom L No darkroom is needed L Darkroom is needed Question 13 Do you use the dazzling protection for the eyepieces _ Yes they are useful L No do not need them no dazzling _ No would need them but they are not comfortable convenient Question 14 Did you have any technical problems with your microscope until now repair replacement L Yes describe L No Part IV Application questions Question 1 In your daily work do you plan to switch between brightfield and fluorescence contrast using just the Primo Star iLED microscope or would you rather use the iLED for fluorescence detection only and a second microscope for brightfield detection Ziehl Neelsen C I would use the Primo Star iLED for both fluorescence and brightfield and would switch between the two modes at least once per day C would use the Primo Star iLED for fluorescence only and will use a second microscope for bright light microscopy C do not think a brightfield microscope will be needed in the future anymore for TB detection i e will only use it for fluorescence Question 2 For which applications would you use the Primo Star iLED C for TB detection only C for Malaria or HAT detection only C for various applications such as TB Malaria Blood Cell Counts urine analysis Try
51. escent Microscopy Page 2 of 15 Staff should have appropriate training and have their performance monitored Laboratory equipment The operating manual and cleaning instructions for all equipment must be readily available Dated service records must be kept for all equipment Microscope and balance must be monitored regularly to ensure consistent performance Specimens and request forms Perform microscopy only upon written request of authorized persons Do not allow oral requests without a completed follow up request form Insist on adequately completed request forms and proper labeling of specimens This ensures positive identification of patients Reject specimens that can not be properly identified are leaking or in broken containers Request a repeat specimen Record the date specimens arrive in the laboratory Document on the request form any delays in the delivery of specimens to the laboratory Evaluate the quality of sputum specimens Record and monitor the number of salivary specimens received by the laboratory Keep laboratory request forms separate from specimens Forms that have been contaminated during transportation or otherwise by specimens should be discarded either by autoclaving or burning Accurately make duplicate form from the original form before discarding Reagents and stains All staining reagents should be labeled with the name date of preparation and date first opened If staining reagents are prepar
52. ext fields where applicable If you complete this form electronically check fields by double clicking on the selected box and by selecting checked For text fields double click on the field and enter default text Please send completed forms to FIND Study Coordinator either by fax 41 22710 0599 or via e mail catharina boehme finddiagnostics org Part I Installation and first use Question 1 Was the installation first use of Primo Star iLED by a microscopist C Self explanatory can be done without reading the user manual L Easy but a user manual with instructions is required C Rather difficult some problems were faced during installation first use L Very difficult cannot be expected of a microscopist Describe difficulties that have occurred or may occur during installation Question 2 Was the installation first use of the battery pack by a microscopist L Self explanatory can be done without reading the user manual C Easy but a package insert is required L Rather difficult some problems were faced during installation first use L Very difficult cannot be expected of a microscopist Describe difficulties that have occurred or may occur during installation first use Question 3 How satisfied were you with the Primo Star iLED user manual _ Easy to read and understand covers all questions had during installation use C Most sections easy to read and understand with some weaknesses in sect
53. field average on average on average 3 210 AFB 1 HPF on 2100 AFB 1 field gt 50 AFB 1 field average on average on average The table below provides the number of fields to be read before providing results using different magnifications of FM Table 2 Magnification of FM objectives and minimum number of fields to be read for FM results using the Primo Star iLED during the project FM objective magnification power 40x Minimum number of fields for FM results Negative 40 Scanty 1 40 40 2 3 20 8 For the purpose of uniform examination and quantitative reporting of results a method has been suggested ref 1 WHO Manual on Microscopy Part II whereby the number of acid fast bacilli observed under fluorochrome staining could be divided by a magnification correction factor to yield an approximate number that might be observed if the same smear were examined under 1000x after carbol fuchsin stain The magnification correction factors for the two FM objectives used in this study are given in Table 3 below Module 8 Reading Recording and Reporting of Fluorescent Smears Page 4 of 12 Table 3 Magnification correction factor FM objective magnification power Magnification correction factor 20x 10 40x 5 Essential Elements of Recordkeeping Accurate recordkeeping in the TB laboratory is essential Recording means keeping the register up to date Lives depend upon it and so does the proper management of the tub
54. he amount of light from light source Light source in the base of the microscope stand Focusing knob that allows a coarse adjustment of the image Focusing knob that allows a fine adjustment of the image Controls the power supply to microscope Controls the amount of voltage supplied to the lamp Used to move the slide in x and y direction for complete coverage of object in our case it is the smear UNPACKING AND USE OF PRIMO STAR iLED For details see User Manual The Primo Star iLED consists of the following parts 1 Preassembled microscope with mounted fluorescence unit binocular tube power supply and transmitted light slider N A 4 Accessory kit 3 Eye cups 2 Battery pack The microscope should be placed on a stable level bench well away from the staining area The battery pack is setup as follows Remove the power supply and its plug from the microscope and connect the battery pack as shown in picture 2 By connecting the power supply to the power supply line the battery pack will automatically start charging the accumulators While the accumulators are being charged you can work with the microscope which is being supplied by the power supply n case of a power cut the battery pack switches automatically to accumulator mode rae To switch the power supply on push the power on button 4 at the front of the housing The battery pack is equipped with two
55. he re checking is to improve the overall quality of smear microscopy therefore regular and timely feedback to the microscopy center is essential if any improvements in performance are to be expected The preliminary observations feedback and remedial action will often be possible at the end of each sampling period Potential sources of errors are to be investigated during the on site evaluation visits Appropriate corrective actions and or remedial training are to be provided during the next visit by the supervisor Critical components of an accurate and practical re checking system include A sufficient number of randomly selected slides to represent work performed The inclusion of minor errors representing false positive or false negative interpretations of scanty results with major errors the smaller sample size aids implementation and sustainability of rechecking programs A system to provide prompt continual feedback and improvements to the laboratories that are supervised Discrepant results will be resolved by Supra National Reference Center Module 9 Assuring Quality of Fluorescent Microscopy Page 7 of 15 Table 4 Investigation of Errors Pattern of errors Possible causes Suggested investigation steps HFP and HFN Unusable microscope Examine a 3 using that microscope Staining problems poor stains insufficient staining time or heating Check stains and staining procedure Technician cann
56. ials are required to perform the microscopy of AFB smears 1 Primo Star iLED microscope 2 Electric power or power pack 3 Lens paper or fine tissue paper 4 Lens cleaning solution 5 Laboratory register 6 Slide storage boxes 7 Red and blue writing pens Microscope Components Review the components and functions of each part of the Primo Star iLED microscope Module 3 Cleaning the objectives Unlike ZN microscopy immersion oil is not used for reading FM smears hence the objective lenses do not require wiping with lens or fine tissue paper to remove traces of oil Reading the smear Keep stained smears in the dark in a box or folder until reading time and read them as soon as possible since fluorescence fades quickly Make sure that the smear is facing upwards when the slide is placed on the mechanical stage Focus the smear using low power objective 10x or 20X Use the objective 40x objective to systematically examine the smear Scan the stained smear systematically from one side to the other and move back at least one length 40 fields have to be scanned before reporting a negative corresponding to 200 high power fields and taking approximately 2 minutes 40x objective Acid fast bacilli appear bright yellow against the dark background material Module 8 Reading Recording and Reporting of Fluorescent Smears Page 2 of 12 Appearance of AFB in Auramine O Smear Tubercle bacilli are quite variable in
57. ides to expedite drying This can produce dangerous aerosols Fix smears by flaming only after they have dried completely SAFE DISPOSAL OF INFECTIOUS WASTE After smears have been processed place all infected materials including closed sputum containers in a discard bag polyethylene if available Discard applicator sticks into disinfectant containing bin used for smearing immediately after use Since all sputum specimens are considered potentially infectious treat all materials in the procedure as contaminated Discard disinfected specimens by one of the following methods Burning Burying Autoclaving To protect the surrounding population the laboratory must dispose of waste safely Burning waste in an incinerator is usually the most practical way for safe destruction of laboratory waste If safe burning can not be arranged discard the waste in a deep pit of at least 1 5 meter depth If an autoclave is available place infected materials inside and follow procedures for safe and adequate sterilization CHEMICAL SAFETY AFB microscopy requires the use of several hazardous chemicals These include concentrated acids alcohols and phenol Take the following precautions when working with chemicals in the TB microscopy laboratory Always wear laboratory coats gloves and safety glasses when handling strong acids Take particular care in diluting concentrated acids ALWAYS ADD THE CONCENTRATED ACID TO WATER This avoi
58. ies Lancet 2006 367 942 43 2 Cambanis A Ramsay A Wirkom V Tata E Cuevas LE 2007 Investing time in mircroscopy an opportunity to optimize smear based case detection of tuberculosis Int J Tuberc Lung Dis 11 40 45 3 Hagemann PKH Floureszenzmikroskopische untersuchungen uber virus und andere microben Zentralbl Bakteriol 1937 140 184 4 Prasanthi K Kumari AR Efficacy of fluorochrome stain in the diagnosis of pulmonary tuberculosis co infected with HIV Indian J Med Microbiol 2005 23 179 85 5 Kivihya Ndugga LE van Claaff MR GithuiWA et al A comprehensive comparison of Ziehl Neelsen and fluorescence microscopy for the diagnosis of tuberculosis in a resource poor urban setting Int J Tuberc Lung Dis 2003 7 1163 71 6 Singh NP Parija SC The value of fluorescence microscopy of auramine stained sputum smears for the diagnosis of pulmonary tuberculosis Southeast Asian J Trop Med Public Health 1998 29 860 3 7 Githui W Kitui F Juma ES et a A comparative study on the reliability of fluorescence microscopy and Ziehl Neelsen method in the diagnosis of pulmonary tuberculosis East Afr Med J 1993 70 263 6 8 Hanscheid T Ribeiro CM Shapiro HM Perlmutter NG Fluorescence microscopy for tuberculosis diagnosis Lancet Infect Dis 2007 7 236 7 9 Steingart KR Henry M Ng V et al Fluorescence versus conventional sputum smear microscopy for tuberculosis a systematic review Lancet Infect Dis 2006 6 570 81 10 Internati
59. ified Types of Errors It is important to re emphasize that random blinded rechecking RBRC is not a method for validating individual patient diagnosis but rather of assessing overall laboratory performance detecting unacceptable levels of errors so that corrective action can be taken and providing continuous motivation for good performance For the purposes of EQA the types of errors are classified on the basis of expected laboratory performance Table 3 not on the potential impact of patient management The monthly sample size has been rounded off to the next higher number and annually adds up to equal or more than the annual sample size Module 9 Assuring Quality of Fluorescent Microscopy Page 6 of 15 Table 3 Classification of Errors Result by MC Result of Controllers pe ee eee te ee Ts fees mT on oe Correct Correct Correct Correct Correct Correct Correct Correct No errors QE Quantification error Minor error LFN Low False Negative Minor error LFP Low False Positive Minor error HFN High False Negative Major error HFP High False Positive Major error Investigation of errors Any error that is detected during rechecking should be investigated by identifying the probable causes based on the results of the checklist and possible corrective actions are suggested Table 4 gives some of the causes and corrective actions for the errors identified Feedback The primary purpose of t
60. in another control smear making sure that the procedure is correct If this gives a good result use this lot to stain routine smears If it does not use a new lot of staining reagent to re stain them Make sure that the new lot has gone through proper quality control Module 6 Preparation of FM Reagents Page 5 of 11 Key messages y g Use quality reagents to prepare staining reagents GS Accurate preparation of reagents is critical to obtain optimum A staining results Quality control of staining reagents using control smears ensures proper performance of newly prepared staining solutions Record quality control results in logbook for quality control of staining reagents Store prepared reagents in clean bottles and out of direct sunlight Module 6 Preparation of FM Reagents Page 6 of 11 Review Module 6 Please answer the following questions based on the preparation of FM reagents module Why must quality reagents be used to prepare staining reagents Why is correct preparation of reagents necessary to obtain optimum staining results What is the role of control smears in evaluating the performance of newly prepared staining solutions How should reagents be labelled and stored Module 6 Preparation of FM Reagents Page 7 of 11 Appendix 1 Worksheet for Preparation of Auramine O Staining Reagents Laboratory Practical Session 1 Item Lot number Actual amount taken Auramine O Aur
61. ing positive controls FM 40 x objective no 40 fields no 40 fields Control Slide AFB color AFB number Decolorization Decision Note Batches checked on Au O batch Hydrochloric acid batch Potassium permanganate batch Methylene blue batch if used Average grading positive controls LED 40 x objective no 40 fields no 40 fields Control Slide AFB color AFB number Decolorization Decision Note Module 6 Preparation of FM Reagents Page 10 of 11 Appendix 3 Recording Worksheet for Laboratory Practical Session 2 Batches checked on Auramine O date prepared Concentration Hydrochloric acid date prepared Concentration Potassium permanganate date prepared Concentration Average grading positive controls AFB 40 fields Control slide ID AFB color AFB No Background Remarks Module 6 Preparation of FM Reagents Page 11 of 11 Module 7 Smear Preparation and Fluorescence based Staining Methods To provide an understanding of proper smear preparation and Purpose staining technique for AFB by fluorescence smear microscopy Prerequisite Module 6 Modules Learning At the end of this module you will be able to OPiectives Safely prepare sputum smears Prepare good quality sputum smears Identify
62. ions Missing topics C Rather cumbersome to read information required is not found easily not enough pictures that allow understanding at first glance weaknesses especially in the following sections Missing topics Comments Part Il Training Question 1a For a microscopist trained in ZN microscopy how intensive should the training for Primo Star iLED be days Question 1b For someone without prior training in smear microscopy how intensive should the training for Primo Star iLED be days Comments Question 2 only to be completed by supervisory sites in phase II How satisfied were you with the Primo Star iLED training manual C Can be used by NTPs for implementation of LED microscopy without major changes C Can be used by NTPs for implementation of LED microscopy but requires some major changes L Requires complete revision Suggestions for changes Part Ill Optics and Handling Question 1 How satisfied are you with contrast color intensity and signal to noise background ratio of Primo Star iLED C Very satisfied better than for the available light microscope enter brand and model of microscope and where applicable fluorescence microscope C Satisfied comparable to available light microscope and where applicable fluorescence microscope C Not satisfied not as good compared to those of the available light microscope and where applicable fluorescence microscope Comments Question 2 How
63. ir lids at all times except when removing specimen for smear preparation Open sputum containers with care and away from the face near the spirit lamp flame Gently open the sputum container especially if the lid clicks or snaps on Do not forcefully shake or stir the sputum in the container Move slowly and carefully while sampling sputum particles and smearing onto slide Avoid any rapid motion when making the smear as infectious aerosols may be produced Safety practices during procedures Disinfect the work area before and after smear preparation Immediately cover any sputum spills with disinfectant before cleaning up the area A phenolic or freshly prepared hypochlorite disinfectant is sufficient Where available use disposable wooden sticks for smear preparation Discard into a receptacle immediately after use If wire loops are used remove residual sputum on the wire loop before flaming Do so by inserting the wire loop in a sand alcohol flask and then moving it up and down or rotating it Never put a wire loop into a flame when sputum is still attached to it as sputum particles containing live AFB will produce infectious aerosols Always keep discard receptacles containing disinfectant in the immediate area Module 4 Safety Precautions for TB Microscopy Page 5 of 13 After sputum is smeared onto the slide let the slide air dry for 15 20 minutes Wet slides can produce aerosols if disturbed Do not flame sl
64. ire a NTP policy choice These include Device used to make smear metal wire or wooden applicators For metal wire loop Nichrome wire 1 meter per year Wire loop holder 1 per 5 years Sand bath 1 per 5 years Spirit lamp 1 per 10 years For wooden applicators disposable bamboo coconut or wooden sticks 1 piece of 10 12 cm in length per smear Glass slides 1 slide per smear For unfrosted slides high quality diamond stylus 1 per technician for 5 years For frosted slides lead pencils of HB grade are sufficient Lens cleaning solutions Refer to microscope manual for specific cleaning solution The following is a list of reagents required to prepare the staining solutions for Auramine staining method 0 1 Auramine Ingredients Quantity per liter 1 Auramine O certified grade 1 0g 2 Alcohol denaturated ethanol or methanol technical grade 100 0 ml 3 Phenol crystals analytical grade 30 0 g 4 Distilled or purified water 870 0 ml To be used for lt 1 month Decolorizing solution 0 5 Acid Alcohol Ingredients Quantity per liter 1 Hydrochloric acid technical grade 0 5 ml 2 Ethanol 100 ml To be used for lt 6 months 0 5 Potassium permanganate Ingredients Quantity 1 Potassium permanganate certified grade 5 0g 2 Distilled water 1000 0 ml To be used for lt 1 month To be used for lt 3 months Module 5 Managing Supplies Page 3 of 11 RECEIVING AND STORING SUPPLIES When
65. isms due to storage in a high humidity environment Penetration of immersion oil between the lenses of the objective through damaged lens cement due to use of poor quality oil such as cedar oil or misuse of xylene this is most likely the cause if a completely hazy field becomes clear after changing the objective A damaged objective due to careless focusing dropping rough changing of sides Frequently encountered operational errors include the following Focusing the first slide using the 100x immersion objective without first passing through a low power Changing slides from under the immersion objective without turning it away first Wiping lenses without first blowing away dust and sand Cleaning lenses or other parts with xylene Using cedar wood oil liquid paraffin or xylene diluted oil instead of pure synthetic immersion oil Keeping the microscope in a confined space and without ventilation in a humid climate LOGBOOK A microscope logbook should be maintained to enter problems encountered in the operation of microscope maintenance schedule repairs done etc FREQUENTLY USED TECHNICAL TERMS IN MICROSCOPY The following terms are frequently used when judging the quality of the optics of a microscope At the end of this training a user appraisal questionnaire will have to be completed for which these terms will have to be understood Technical Term Definition Contrast The difference in brightness between the light a
66. l burden which in most instances is specific enough that no confirmatory testing is needed In many countries it is based only on the examination results of Ziehl Neelsen ZN stained smears However only tiny amounts of material are examined as little as 0 2 micro L and hence bacteria must be present in high concentrations to be visible typically over 10 000 acid fast bacilli per mL Before declaring a smear as negative a minimum of 100 microscopic fields have to be examined under 100 x oil immersion objective which takes about 3 to 5 minutes of a technician s time In busy overburdened laboratories smears may not be examined for the recommended amount of time resulting in lower sensitivity A re examination for longer periods proved that the negative smears were in fact positive Since the first description of the auramine O fluorescence microscopy technique by Hagemann 3 in 1937 numerous reports have confirmed the superior diagnostic performance of fluorescence microscopy compared with Ziehl Neelsen ZN staining and light microscopy 4 8 In a systematic review of 18 studies Steingart et al 9 reported that fluorescence microscopy of auramine stained smears provides similar specificity and increased sensitivity mean improvement of 10 compared with light microscopy of ZN stained smears In addition to increased sensitivity fluorescence microscopy also allows more rapid screening of sputum smear specimens From an o
67. lean grease free unscratched slides which are free from fingerprints Using a pencil record the laboratory register serial number and order number of the sputum specimen on the frosted end of the slide If plain unfrosted slides have to be used labeling is best done using a diamond pencil Ensure that the number on each slide corresponds to the number on the specimen container Module 7 Smear Preparation and FM Staining Methods Page 2 of 14 2 Sputum smearing Using the end of an applicator stick or wire loop select and pick up the blood specked opaque grayish or yellowish purulent parts of sputum Prepare the smear in an oval shape in the center of the slide Figure 1 The smear size should be about 3 cm in length x 2 cm wide which will allow 100 150 fields to be counted in one length For good spreading of sputum firmly press the stick perpendicular to the slide and move in small concentric circles or coil like patterns Place the used stick into a waste receptacle which also contains a disinfectant Use a separate stick for each specimen Alternatively if a wire loop is used instead of a broken stick dip the wire loop into a sand alcohol bottle Figure 2 Remove the excess sputum from the wire loop by moving it up and down After each smear is completed heat the wire loop in a flame until red hot Thorough spreading of the sputum is very important it should be neither too thick nor too thin Prior to staining hol
68. lood slides with filtered 0 1 Auramine O solution Do not heat Keep the staining reagent for at least 20 minutes Make sure that the smear area is continuously covered with Auramine solution by adding more if needed Rinse with water and drain Apply decolorizing solution 0 5 Acid alcohol for 3 minutes Gently rinse with water until the macroscopically visible stain has been washed away and drained Flood smear with 0 5 Potassium permanganate solution for 1 minute Time is critical because counterstaining for a longer time may quench the acid fast bacilli fluorescence Gently rinse with water and drain Air dry on a slide rack The staining procedure steps 1 7 is shown graphically in the next pages Module 7 Smear Preparation and FM Staining Methods Page 6 of 14 STAINING PROCEDURE Step 1 Place slides 1 cm apart on a staining rack with the smeared side facing up Step 2 Flood the slides with freshly filtered auramine phenol Let stand for 20 minutes SPECIMEN SPECIMEN _ SPECIMEN SPECIMEN Module 7 Smear Preparation and FM Staining Methods Page 7 of 14 Step 3 Rinse well with running water taking care to control the flow so as not to wash away the smear Step 4 Decolorize by covering completely with acid alcohol solution for 3 minutes Module 7 Smear Preparation and FM Staining Methods Page 8 of 14 Step 5 As before rinse well wi
69. microscopy centre Patient referral Ideally you can refer a patient to the microscopy centre so that a specimen can be collected under supervision If an unsatisfactory specimen is submitted then a repeat sample can be obtained immediately The disadvantage of this option is that the patient may find it expensive or impractical to travel to the microscopy center if it is in a different location from the clinic Patients may be reluctant to seek help and diagnosis may be delayed Specimen referral Alternatively the peripheral health center can supervise the patient in collecting an appropriate specimen which is then forwarded to a microscopy center Transport specimens once or twice each week although in some remote settings this may not always be possible To prevent leaks and breakage place specimens carefully in the specimen container Clearly label each specimen with the patient identification and include a completed request for sputum examination Slide referral Time delays for slide referrals may occur Training and periodic supervision is required to assess the quality of smear preparation There are however several advantages Heat fixed sputum smears are less infectious than sputum specimens are and require less packaging for transport ASSESSING SPECIMEN QUALITY Upon arrival in the laboratory assess the quality of samples TB sputum can have various colours and aspects If the sample is liquid and as clear as water without
70. nd dark areas of a picture Color intensity Brightness brilliance and saturation of colors Signal to noise ratio Compares the level of a desired signal AFBs to the level of background noise Homogeneity of Homogeneous illumination of the image with light that is fluorescence illumination bright glare free and evenly dispersed in the field of view Resolution The smallest distance between two points on a specimen that can still be distinguished as two separate entities The resolving power of a microscope is the most important feature of the optical system and influences the ability to distinguish between fine details of a particular specimen Depths of focus The range over which the image plane can be moved while an acceptable amount of sharpness is maintained Appendix 1 Specifications for a LED based fluorescence microscope e Binocular microscope for use with electric light via power line or alternatively via battery pack Battery pack can also be used as uninterruptible power supply and is usually included as an accessory e Observation tube binocular 30 deg inclination viewing angle and 360 deg rotation e Stage rectangular built in mechanical stage with vernier scale minimum 14 mm x 135mm No polymer belts metal cables timing belt systems or non metallic components are acceptable in the drive mechanism Coaxial controls must be low mounted for ease of use Stage finger assembly is to be slide friendly so that it does not
71. ne Purpose Learning Objectives Content Outline Handout and Exercises To provide you with an overview of the LED demonstration project phases and roles and your responsibilities during the project At the end of this module you will be able to Explain the LED demonstration project phases and the study flow during these phases Refer to the correct documents to obtain further details LED demonstration project phases Study documents Handout SOP and Protocol for LED Demonstration Project WHAT IS A DEMONSTRATION PROJECT Demonstration projects are carried out in the context of routine clinical services provision either directly by the National TB Program NTP or by other agencies working in collaboration with the NTP These are large studies with 10 000 or more patients enrolled are intended to provide the evidence that new tests that perform well in controlled settings can also have an important medical and public health impact when implemented in programmatic settings Endpoints commonly studied include feasibility of assay implementation comparative cost of the new versus the old technology and impact on speed or accuracy of detection and subsequent patient management The results of demonstration projects are compared against Customer Requirements as stated by Ministries of Health WHO and other international technical agencies donors and patients Data from Demonstration projects are compiled analyzed
72. ng Quality Improvement QI A process by which the components of smear microscopy diagnostic services are analyzed with the aim to identify and permanently correct any deficiencies Data collection data analysis and creative problem solving are skills used in this process QUALITY CONTROL IN THE TB LABORATORY Quality Control helps to ensure that the results produced by a laboratory are accurate reliable and reproducible The QC program should be performed regularly and to be effective the process must be practical and readily included in standard laboratory reporting practices All laboratory technicians are responsible for performing recording and reporting results of QC Many components of QC are either performed in conjunction with routine testing or as part of the regular management of the laboratory yoratory arrangement and administration Ensure that doors into the laboratory are always closed Work areas equipment and supplies should be arranged for logical and efficient workflow Work areas should be kept free of dust Benches should be cleaned at least daily with an appropriate disinfectant Use laboratory procedures that comply with NTP guidelines Every procedure performed in the laboratory must be performed as per the SOP The procedures must be kept in the laboratory and be readily available Any changes to procedures must be dated and initialed by the laboratory supervisor Module 9 Assuring Quality of Fluor
73. ng a negative result with Primo Star iLED Key points for maintenance Storage and cleaning Review Module 3 Please answer the following questions based on the use and maintenance module What would be possible reasons to switch from ZN microscopy to LED based fluorescence microscopy If the fluorescence contrast is reduced when using Primo Star iLED what is the most likely cause What entries are made in the Logbook When and how are microscope objectives cleaned Module 4 Safety Precautions for TB Microscopy Including Collection and Transport of Sputum Samples Purpose Prerequisite Modules Learning Objectives Content Outline Handout and Exercises Appendix To provide you with an understanding of safe handling techniques and precautions while performing AFB smear microscopy with the knowledge and skills for proper collection and transport of sputum samples for AFB microscopy None At the end of this module you will be able to Explain airborne transmission of TB Describe risks involved when collecting sputum Describe personal health and safety practices Describe why there should be three distinct areas in the TB laboratory Describe methods for the disposal of contaminated material Describe chemical safety precautions in the laboratory Describe specifications of suitable containers for sputum collection Explain the collection strategy spot morning spot Describe and demons
74. nsider re staining for rechecking Assess concentration of phenol basic fuchsin Auramine O potassium permanganate and methylene blue Poor smearing technique Test stain with known negative smears Problems with microscope Check microscope with known positive smear Careless microscopy Exclude other causes Very high proportion LFN Reading error Less than recommended fields are being read probably due to high workload or inadequate training Concentrated Methylene blue for ZN method Check the concentration of Methylene blue Many QE too low grading Poor staining Check the concentrations of basic fuchsin in ZN stain Auramine O in FM stain and phenol in both Problems with microscope Check microscope with known positive smear Module 9 Assuring Quality of Fluorescent Microscopy Page 8 of 15 Responsibilities of the Laboratory Technician 1 Store all slides in a way that allows retrieval of every slide identified for the rechecking sample when 100 re checking is not required They must be stored in the slide boxes in the same order as they are listed in the laboratory register 2 Label slides in a manner consistent with the laboratory register to ensure that the correct slide can be matched to the result The labeling must be legible The result of the smear examination must not appear on the slide 3 Always store slides in closed boxes away from direct sunlight Thi
75. nt of disease is the most significant predictor of the infectiousness of a patient Household contacts of smear positive patients have tuberculin positivity rates of 30 50 compared with contacts of smear negative patients who have tuberculin positivity rates of only about 5 The infectiousness of the patient may also depend on how often that person coughs Coughing is a good mechanism for producing droplet nuclei and a higher prevalence of tuberculin reactivity has been reported among contacts of frequent coughers i e people who cough gt 48 times per night than among contacts of infrequent coughers i e people who cough lt 12 times per night Interestingly singing produces infectious droplet nuclei as effectively as coughing and several outbreaks in choirs have confirmed that singing can spread infection However while coughing and singing may increase the contagiousness of a patient the radiologic extent of disease and smear status remain the best indices of infectivity Module 4 Safety Precautions for TB Microscopy Page 2 of 13 Proper Collection of Sputum Collecting sputum represents the greatest hazard to a laboratory technician because infectious aerosols may be produced by coughing When patients come coughing into the laboratory ask them to cover their mouth Wherever possible collect specimens outside where air movement will rapidly dilute infectious droplets and UV rays from the sun will rapidly inactivate TB bacilli
76. ommended to use the WHO or IUATLD laboratory register as a guideline The format of this register should never be altered by laboratory staff This is a record book maintained by the technician technologist responsible for sputum smear examination of patients with suspected TB and their follow up examinations The TB laboratory register must include the following data for each patient with suspected TB The laboratory register should include e Laboratory serial number e TB registration number e Date the specimen was received e Patient s name sex age and address e Reason for exam diagnosis or follow up e Smear results e Signature of person responsible for tests Module 8 Reading Recording and Reporting of Fluorescent Smears Page 6 of 12 Make sure all necessary columns are filled in However if patients with suspected TB do not deliver three sputa in those settings where three sputa are required according to NTP guidelines leave the data field blank A blank space is not a negative result Results must be accurate scanty results should be entered as such and not changed to negative or positive A positive follow up result must also be registered accurately whatever the conversion or cure targets may be Reset the laboratory register number to one on January first each year DO NOT reset at the end of each day week or month Enter patients successively increasing the line number by one each time The line number is sufficien
77. on and Fluorescence based Staining Methods Reading Recording and Reporting of fluorescent smears Assuring Quality of Fluorescence based AFB Microscopy Module 1 Introduction The Possible Role of LED based Fluorescence Microscopy in Improving the Global Tuberculosis Situation Workshop goal Certification Criteria This workshop is intended for microscopists and supervisors participating in the LED demonstration projects Training participants will already have experience in ZN microscopy and or conventional fluorescence microscopy During this workshop you will gain the knowledge and skills to perform fluorescence based smear microscopy using the Primo Star iLED accurately and reliably in a safe timely and professional manner You will be awarded a certificate upon successfully completing the Fluorescence based AFB Microscopy Training Workshop after you have Attended and actively participated in all theoretical and practical sessions Successfully reported the AFB smear results by completing EQA panel test examination Introduction The diagnostic technology recommended in current control strategies is sputum microscopy which was developed in the 1880s and has remained essentially unchanged since then Smear microscopy is an attractive technology for public health programmes as it requires simple equipment only can be used for more than one purpose and provides visual evidence not only of tuberculosis but of bacteria
78. onal Union Against Tuberculosis and Lung Disease IUATLD technical guide sputum examination for tuberculosis by direct microscopy in low income countries 5th ed Paris IUATLD 2000 11 Cambanis A Ramsay A Wirkom V Tata E Cuevas LE Investing time in microscopy an opportunity to optimise smear based case detection of tuberculosis Int J Tuberc Lung Dis 2007 11 40 5 12 Bennedsen J Larson SO Examination for tubercle bacilli by fluorescence microscopy Scand J Respir Dis 1966 47 114 20 13 Anthony RM Kolk AH Kuijper S Klatser PR Light emitting diodes for auramine O fluorescence microscopic screening of Mycobacterium tuberculosis Int J Tuberc Lung Dis 2006 10 1060 2 14 Hung NV Sy DH Anthony RM Cobelens FGJ van Soolingen D Fluorescence microscopy for tuberculosis diagnosis Lancet Infect Dis 2007 7 238 9 15 Zeiss 7 November 2007 The Foundation for Innovative New Diagnostics FIND and Carl Zeiss Announce Collaboration to Develop an Affordable Fluorescence Microscope for the Diagnoses of Tuberculosis and Other Infectious Diseases in High Burden Countries http www zeiss de c12567be0045acf1 Contents Frame 520bdbfaeb127b00c125738d0033b52c 16 Fujiki A AFB Microscopy Training Tokyo Japan The Research Institute of Tuberculosis 2005 17 Central TB Division DGHS MoHFW Government of India New Delhi 110011 Manual for sputum smear Fluorescence microscopy Module 2 Demonstration Project Primostar iLED Study Outli
79. onstration project calculations for the most critical supplies will be made by the supervisory site with the help of an Excel spreadsheet provided by FIND see below The supervisory sites only need to enter the number of smear exams performed at a microscopy centre per month and where applicable the stock on hand at the supervisory and for general supplies only at the microscopy sites The spreadsheet provides an accurate estimate of supplies actually required for one quarter by calculating the quantity of each item for one quarter of operation plus a reserve quantity of 20 Depending on how reliable the ordering system is and how quickly placed orders are received the reserve quantity can be increased or decreased From that estimate general supplies you already have on hand you should know these numbers from your physical inventory i e stock count are subtracted automatically The result should be the amount of items you must order to insure uninterrupted testing during the next quarter of operation Module 5 Managing Supplies Page 5 of 11 Quarterly Management of Supplies for iLED Demonstration Project Number of slides per month TOTAL Enter Product Example Merck 101301 Au O C I 41000 50 g bottle 516724 Phenol 500 g Staining solution requirements slide See below Auramine
80. ontaining 5 phenol or 0 5 sodium hypochlorite solution Hand washing and careful techniques are mandatory for safe laboratory practice in all countries Laboratory Coats Laboratory coats are not required when assisting in specimen collection or performing sputum microscopy A lack of laboratory coats does NOT mean that sputum microscopy cannot be performed If they are available laboratory coats of various sizes should be provided and cleaned by the laboratory organisation They should be tied at the back not the front and be made from water resistant materials to avoid liquids soaking into the gown Laboratory coats must NOT be worn outside of the laboratory Masks One of the greatest false beliefs is that a standard surgical mask will protect the wearer from becoming infected with TB These masks are made from porous material that will not trap TB bacilli and have an extremely poor fit leaving large gaps between the face and mask N95 duck bill respirators often incorrectly referred to as masks and particulate respirators are expensive and are not necessary for laboratory technicians carrying out sputum smear preparations only Such equipment must be selected and fitted correctly to be functional Appropriate Disinfectants Phenolic agents 5 phenol in water or a phenolic disinfectant product diluted as per label are excellent disinfectants for cleaning up sputum spills and for decontaminating equipment and single use
81. oscopy is not used more widely is the need for a more complex and expensive fluorescent microscope the limited lifespan typically 200 300 hrs and the high cost of the short arc mercury vapor lamp MVP which has traditionally been used as the excitatory light source Repeated on and off switching as may occur with unreliable local power supply shortens the lifespan even further 13 In addition MVPs are energy inefficient and require an extensive power supply they may also fail catastrophically and release toxic mercury into the environment 13 Consequently fluorescence microscopes provided by donor agencies often fall into disuse because of high maintenance costs 14 Light emitting diode LED technology provides a cheap and reliable light source with a usable lifespan of 150 000 h repeated on and off switching does not reduce its usable lifespan and it does not pose a potential toxicity risk 13 Initial studies indicated that LED fluorescence microscopy with use of a royal blue LED light offers a valid alternative to the MVP 13 14 but data regarding its diagnostic use or operational impact remain limited A new generation of fluorescence microscopes has now been developed based on LED technology A leading microscope manufacturer Zeiss Microlmaging G ttingen Germany in a joint development agreement with FIND has developed a fluorescence microscope Primo Star iLED 15 Other LED based approaches such as the FRAEN
82. ose the containers the need for collecting real sputum not saliva how to produce good sputum i e by repeated deep inhalation and exhalation of breath followed by cough from as deep inside the chest as possible how to avoid contaminating the exterior of the container i e by carefully spitting and closing the container how to collect and safely deliver the morning sputum to the laboratory and the need for three sputa to facilitate diagnosis Module 4 Safety Precautions for TB Microscopy Page 9 of 13 A good specimen should be approximately 3 5 ml It is usually thick and mucoid It may be fluid and contain pieces of purulent material Color varies from opaque white to green Bloody specimens will appear reddish or brown Clear saliva or nasal discharge is not suitable as a TB specimen SPECIMEN HANDLING AND REFERRAL Specimen handling For optimum patient management process the specimen as soon as possible i e lt 24 hours For microscopic examination the interval between collection and staining matters little Acceptable results can be obtained even on delayed specimens If the peripheral health centre does not perform microscopy there are several options Each has advantages and disadvantages Depending on local circumstances one or more options may apply Refer the patient to a health centre where microscopy is performed Collect a sputum specimen in a leak proof sputum container and refer it to the
83. ot recognize AFB Test with clear cut positive amp negative slides and good microscope Gross neglect overworked lack motivation Exclude other causes HFP with or without LFP Administrative error Compare lab register and verify correct slide number and result Exclude causes of more frequent HFP such as low concentration of sulphuric acid unusable microscope untrained or inexperienced LTs Poor registration routine Check accuracy of lab register and other record keeping Staining problems Fading Check stains and staining procedure consider re staining for rechecking Assess concentration of phenol basic fuchsin Auramine O and methylene blue Technician unclear on AFB appearance Look for inconsistent results of suspects regularly single pos low positive in lab register Many LFP with or without occasional HFP Problem with controllers Technician unclear on AFB appearance Contaminated stain reagents Evaluate controllers Recheck sample of LFP from laboratory register Test stain with known negative smears check the distilled water used for stain preparation HEN with or without LFN Administrative error Compare lab register with QC listing correct slide number amp result Very thick smears and or poor light Evaluate quality of smear preparation check microscope Gross neglect Exclude other causes Staining problems Check stains and staining procedure co
84. owing questions based on the safety precautions module How is TB transmitted from person to person What are appropriate laboratory disinfectants What precautions must you take when handling specimens Why do surgical masks offer little protection against TB What precautions should you take when preparing dilutions of strong acid Module 4 Safety Precautions for TB Microscopy Page 12 of 13 Review Module 4 Please answer the following questions based on the safety precautions module What is the benefit of an early morning specimen Why should sputum never be collected in the laboratory What are the important instructions that should be given to patients for the collection of good quality sputum specimens How should salivary specimens be handled in the laboratory Module 4 Safety Precautions for TB Microscopy Page 13 of 13 Module 5 Managing Supplies for Fluorescence based AFB Microscopy Purpose Prerequisite Modules Learning Objectives Content Outline Handout and Exercises Appendix To provide you with an understanding of inventory and to help you calculate your laboratory supplies for fluorescence microscopy for a given period None At the end of this module the participant will be able to List supplies required to perform fluorescence based smear microscopy Calculate supplies required by completing Excel sheet Order supplies Maintain proper records Explain use of sto
85. panosomiasis Question 3 Do you see a significant gain in speed when reading slides with Primo Star iLED 30 fields compared to ZN 100 fields C Yes LI Yes for negative and low positive slides only L No Question 4 If you had to decide whether to change a majority of microscopy centers in your country from light microscopy to LED based fluorescence microscopy would you recommend to the Head of the National Health Program to switch to LED L Yes Reasons C In principle yes But would prefer using another microscope and not the Primo Star iLED Reasons L Only for low volume microscopy centers Reasons L Only for high volume microscopy centers Reasons L Only in specific settings Define setting Reasons L No Reasons C No But would switch centers that currently have and use a conventional fluorescence microscope to LED fluorescence Reasons Thank you very much for helping us with your feedback K ey messages Familiarize yourself with all working parts of your 7 microscope AS Record all problems with the microscope in a logbook Call for help when troubleshooting any problems related to function Fluorescence microscopy saves time and is more sensitive than ZN microscopy Bulb replacement will very rarely be necessary when using LED based microscopes gt 10 000 h Read at least 30 high power fields 20x for FM smears and 100 high power fields for ZN smears before reporti
86. perational perspective this is highly advantageous particularly when high numbers of samples are screened per day because the majority of laboratory time is spent confirming negative smear results According to the International Union Against Tuberculosis and Lung Disease technical guidelines for sputum microscopy at least 5 minutes of screening time is required to correctly identify a negative smear result when conventional light microscopy is used 10 However under routine field conditions the time spent per slide is often far less than the minimum required An operational study from Cameroon demonstrated a median sputum microscopy examination time of only 2 min 11 Almost 50 of the cases detected after a thorough 10 min evaluation were missed during routine investigation 11 which demonstrates the negative impact that conventional light microscopy may have on early case detection and diagnostic delay A comparative study reported that a mean time of 1 min to examine a sputum smear with fluorescence microscopy achieved higher sensitivity and equivalent specificity than did conventional light microscopy with an examination time of 4 min 12 The auramine O stain is inexpensive and the procedure is easier and quicker than ZN staining Despite the clear operational advantages of fluorescence microcopy conventional light microscopy remains the most widely used diagnostic test in resource limited settings The main reason that fluorescence micr
87. problems with smear preparation Perform the Auramine O staining of sputum smears Troubleshoot problems with the Auramine O method Content Outline Labeling of slides Selecting the best portion of the specimen for smear preparation Techniques for preparing smears Principles of the Auramine O method The Auramine O staining procedure Handouts and Laboratory Practical Session 3 Preparation of smears Exercises Laboratory Practical session 4 Staining sputum smears Appendix None Module 7 Smear Preparation and FM Staining Methods Page 1 of 14 Module 7 Smear Preparation and Fluorescence Microscopy Staining Methods The quality of work in AFB diagnostic microscopy depends on a number of factors like specimen collection quality of reagent staining technique reading of smear reporting and recording and a technician s level of training However collecting a suitable specimen and making a good smear are critical as the quality of the rest of the procedure depends on it Smear preparation must be performed carefully and with attention to detail Overview of smear preparation 1 Label each slide with the correct number serial and order number 2 Smear sputum onto slide 3 Allow smear to air dry 4 Heat fix smear The physical appearance of sputum specimens salivary mucopurulent bloody and muco colloidal watery are shown below PREPARING SPUTUM SMEARS 1 Labeling the slides Select new c
88. r side up Leave enough space between slides to prevent the transfer of material and or staining solution from one smear to another APPLY Auramine stain Cover the entire surface of the slide with filtered Auramine solution If the staining solution drains off add more stain to cover the entire slide DO NOT HEAT Leave for 20 minutes and do not let the solution dry RINSE the slide Tilt the slide to drain off excess stain and then rinse the staining solution off with a gentle stream of water It may be convenient to use a beaker flask or squeeze bottle to pour the water onto the slides When rinsing slides avoid getting water stream directly on the smear vigorous washing may cause the smear to lift Tilt the slide to drain off excess rinse water DECOLORIZE the smear by covering the whole slide with 0 5 hydrochloric acid alcohol solution and leave it for a maximum of 3 minutes WASH the slide again with a gentle stream of water Tilt the slide to drain off excess water QUENCH the smear by covering the entire surface of the slide with potassium permanganate solution and leave it for a maximum of 1 minute DRAIN off the permanganate solution Rinse the slide again with a gentle stream of water Make sure the stained smear is free from stain deposits dirt debris and crystals produced by overheating during staining Underside of the smear should be wiped if possible with alcohol PLACE on the slide rack to air dry Do not allow th
89. re diaphragm of the condenser Knurled knob for vertical adjustment Coarse focusing dial or knob left side Fine focusing dial or knob left side Knurled ring for adjusting the smoothness of the coarse focusing drive Below is a list of the microscope components and their respective functions Microscope components Eyepieces Diopter adjustment ring Binocular tube Nose piece Objective lenses Stage Slide holder Condenser with diaphragm Filter Field diaphragm Lamp Coarse focus knob Fine focus knob Power switch Voltage regulator Stage movement knobs Function Pair of lenses used to view the magnified image from the objective lens Used to focus by turning clockwise or anticlockwise to obtain a sharp image The part holding the eyepieces and dividing the light between them It is used to adjust the distance between the eyes so that a single overlapping image is obtained The mechanical and revolving part that holds the objective lenses Lenses of various magnification power used to view the object Horizontal platform for placing the object for viewing Mechanical arm that is used to hold the object or slide for smooth and uniform movement The lens system that concentrates the light on the object to be magnified It contains an iris diaphragm meant to reduce glare from dispersed light A blue colored glass that makes the light in the visual field to appear as natural daylight Controls t
90. reparation of Fluorescent Microscopy Reagents Reagent preparation requires equipment for weighing and measuring Distilled or purified water free of environmental mycobacteria must also be available Environmental mycobacteria often colonize water tanks and taps and could on rare occasions result in a false positive reading Therefore avoid using tap water Fluorochrome staining employs dyes which fluoresce and stain acid fast organisms The nonspecific background fluorescence of tissue debris may be suppressed by a counterstain such as potassium permanganate Good staining reagents especially those made with a high quality Auramine O phenol dye are essential to detect acid fast bacilli AFB While it is easy to demonstrate AFB in a highly positive smear only a good staining reagent will also be able to show the AFB when they are rare or damaged due to drug treatment and are especially difficult to stain Poor quality staining reagents may not show these AFB and a case of TB maybe missed EQUIPMENT REQUIRED FOR STAINING REAGENT PREPARATION The following list is required for preparing staining reagents A balance or weight scale with a sensitivity of 0 1 gram g Measuring cylinders of 100 mL 500 mL and 1000 mL capacity one each Large Erlenmeyer conical flasks or flat bottomed balloon flasks capacity at least one liter A spirit lamp for heating A stirring plate with heating and magnetic stirrers this is preferable when p
91. reparing larger quantities Containers for the newly prepared staining reagents dark amber glass bottles are recommended but plastic bottles or containers with tight closures may be easier to transport Labels for bottles Brushes to clean bottles before reuse Funnels to fill bottles one funnel for each solution AFB positive and negative unstained control smears REAGENTS REQUIRED FOR STAINING REAGENT PREPARATION Preparation of Fluorescent Microscopy Reagents Auramine O stain For staining reagent preparations using auramine use the following items Auramine O powder of good quality Phenol crystals of good quality The crystals should be almost colorless quality must be assessed through quality control of AFB staining AFB smears should yield solid homogenous and strong red staining bacilli Alcohol can be denatured 95 ethanol or methanol Water distilled or purified Module 6 Preparation of FM Reagents Page 2 of 11 Decolorization solution For staining reagent preparations using acid use the following items Hydrochloric acid 37 fuming Alcohol denatured 95 ethanol or methanol Quenching solution Permanganate Potassium permanganate certified Water distilled or purified Prepare the final solutions according to the following guidelines 1 liter of auramine stain final concentrations 0 1 Auramine O e Disolve 1 g of Auramine O in 100 ml of alcohol denatured ethanol or methanol e Dissolve
92. rescence staining solutions be stored Module 5 Managing Supplies Page 11 of 11 Module 6 Preparation of Reagents for Fluorescence based AFB Microscopy Purpose Prerequisite Modules Learning Objectives Content Outline Handout and Exercises Appendices To provide you with an understanding of staining reagents and their preparation in acid fast staining Module 4 At the end of this module you will be able to Describe the importance of using quality chemicals for reagent preparation Prepare reagents required for Auramine O method Describe the safety requirements for reagent preparation Use positive and negative control slides for the quality control of Auramine O reagents Explain the use and frequency of routine quality control procedures Equipment required for staining reagent preparation Reagents required for the preparation of stains Methods for staining reagent preparation Storage of staining reagents Quality control QC of freshly prepared staining reagents Laboratory Practical Session 1 Reagent preparation Laboratory Practical Session 2 Quality control of staining reagents Appendix 1 Worksheet for preparation of Auramine O staining reagents Appendix 2 Example of logbook for quality control of Auramine O staining reagents Appendix 3 Recording worksheet for Laboratory Practical Session 2 Module 6 Preparation of FM Reagents Page 1 of 11 Module 6 P
93. s procedure is important for FM smears 4 Label the slide box with the name of Demonstration site and date 5 The lab technician if possible in consultation with Lab Head and Medical Director should address the problems identified by the Supervisor OSE Summary Module 9 Assuring Quality of Fluorescent Microscopy Page 9 of 15 K a a a Everybody is responsible for ensuring that laboratory results are accurate and reliable BA S To demonstrate and maintain high quality results a laboratory s performance needs to be monitored by the following regular QA activities e Internal Quality Control QC e External Quality Assessment EQA QC is the process of effective and systematic internal monitoring of routine laboratory work EQA is the systematic and independent assessment of laboratory performance Module 9 Assuring Quality of Fluorescent Microscopy Page 10 of 15 Review Module 9 Please answer the following questions based on the FM quality assurance module What are the components of Quality Assurance What is Quality Control What is blinded slide rechecking For blinded slide rechecking how does the technician store the slides Module 9 Assuring Quality of Fluorescent Microscopy Page 11 of 15 What should you do to make sure that the ZN amp FM reagents are working properly Which method are used for identifying AFB during ZN baseline phase validation phase implementation phase and
94. s see Table 1 for frequency of completed forms retrieval EXTERNAL QUALITY ASSESSMENT EQA AND WHY IT IS IMPORTANT The purpose of EQA is to help laboratories identify errors and improve practices for better performance EQA does not identify individual slide errors nor validate individual patient diagnoses Involvement in an EQA activity should not be seen as a threat but rather as an Most laboratory technicians want to provide accurate testing Good performance in EQA activities reassures them that their results are contributing to TB diagnosis and control opportunity to strengthen skills During this study three methods will be used to evaluate laboratory performance Onsite evaluation Panel testing Blinded smear rechecking Table 1 Evaluation of laboratory performance Study phase ZN Baseline iLED Training Validation Implementation Continuation Module 9 Assuring Quality of Fluorescent Microscopy Frequency of retrieving slides and forms Once every 2 week NA Daily Once every 2 week Once a month Supervisory visit with checklist Once a month NA Once every 2 week Once a month Once a month Panel testing Proficiency Testing 20 slides 10 ZN 10 Auramine Proficiency at the end of this phase determined by analysis of rechecking results and by proficiency panel testing 10 Auramine slides NA slides re checked by supervisory site 100 NA 100
95. t for identification of the request form Slides require an extension to identify the first spot second morning or third spot sputum of a series in those settings where three sputa are required according to NTP guidelines Add a or b or a or b or c if two or three sputa are required after the line number Allocate a separate line and serial number in the register to specimens submitted for follow up examination Always fill in the reason for examination i e diagnosis or follow up Use a tick mark to indicate whether the specimen is diagnostic and indicate the TB number for follow up patients A diagnostic patient with specimens with negative results in all smears will submit specimens after completion of two weeks of antibiotic therapy or according to NTP guidelines and these are labeled as RE Supervisors should analyze the register when conducting a laboratory review as it provides a simple easy and rapid summary of the work conducted in a laboratory and assessment of its performance In positive diagnostic samples it can be helpful to obtain the patient s registration number from the NTP this number should be added either under the tick mark or in the remarks column False negative consequences False negative means that the result reported as negative was actually positive Patients with TB may not be treated resulting in ongoing disease disease transmission or death Intensive phas
96. t received received on hand units requested Module 5 Managing Supplies Page 7 of 11 Quarterly Report Quarter year STOCK SUMMARY FORM CEMlelacscsatviiusssacd DISWICl seser PROGION ewe cesceretts eorne Case Number of suspects Number with at least detection examined during the one positive or oeecteesee quarter scanty smear Stock left at the end of the quarter in liters Slides seen Sputum cups S T Sage 1 Carbol 25 Sulphuric 0 1 fuchsin e acid Il 0 sree S blue 0 5 0 5 0 1 AUO I Hydrochloric Potassium acid I permanganate 1 Module 5 Managing Supplies Page 8 of 11 H Maintain an adequate inventory at all times to ensure fam uninterrupted service Quantity required is estimated based on smears performed reserve stock stock in hand actual order for next Key message quarter Never order more than you require in the next quarter Record and account for all items in the inventory Always inspect a new shipment before accepting it Module 5 Managing Supplies Page 9 of 11 Review Module 5 Please answer the following questions based on the supply management module How do you determine reserve stock level How do you determine the order for the next quarter What procedure should you follow when receiving new supplies Module 5 Managing Supplies Page 11 of 11 How amp for how long should fluo
97. th water so as to wash away the acid alcohol Step 6 Quench with 0 5 potassium permanganate for 1 minute Module 7 Smear Preparation and FM Staining Methods Page 9 of 14 Step 7 Rinse with water and slope the slides to air dry EVALUATING SMEARS Spend time evaluating good and bad smears Without a quality smear the procedure of diagnostic microbiology is seriously impeded Bad smears can lead to false results The quality of examination depends on making good smears In this training the preparation of good smears is a very important process Module 7 Smear Preparation and FM Staining Methods Page 10 of 14 Key messages AS Label slides with the laboratory serial number and specimen number e g 562 2 Select the most purulent portion of the sample for smear preparation The size of the smear should consistently be 2 3 cm by 1 2 cm the smear should be thin enough that you can read newsprint through it Use gentle heat fixation for smears In ZN staining carbol fuchsin is heated to steaming at least twice Leave it on the smear for 10 minutes Do not extend the time for counterstaining with methylene blue In Auramine staining leave the Auramine solution on the smear for 20 minutes DO NOT HEAT AURAMINE SOLUTION Do not extend the time for quenching solution with potassium permanganate Module 7 Smear Preparation and FM Staining Methods Page 8 of 14 Review Module
98. tive 1 sputum Let this sputum stand for one or more days at room temperature to allow the sputum to liquefy Then with the container closed mix the contents carefully and make as many smears as possible from this same low positive sputum Check the average number of AFB by staining a few randomly selected smears from the entire batch Record this number in your staining reagent logbook Ensure that sputum used to prepare negative control smears has been extensively examined to ensure that there is no AFB Prepare smears and fix them To protect fixed unstained smears from dust and sunlight store them in a separate and labeled slide box QUALITY CONTROL OF FRESHLY PREPARED STAINING REAGENTS After preparing staining reagents always perform quality control for each batch of staining reagents prepared Quality control is essential to ensure that the staining reagents work well and that they do not contain artifacts or contaminating AFB It is more efficient to prepare bigger batches if very large flasks are available Module 6 Preparation of FM Reagents Page 4 of 11 Keep accurate records in a logbook for quality control see Appendix 2 This serves as an important reference record to ensure good staining reagents In the logbook identify the batches by name of reagent and preparation date as on the bottle labels Perform QC by using one or more freshly prepared staining reagents and the normal staining procedure as described for posi
99. tive controls Examine all controls carefully for number completeness and intensity of color of AFB as well as color and complete destaining of background Record the results in logbook for quality control of staining reagents as in the example given later in the document If unsatisfactory results are obtained in the staining of AFB Auramine O and other reagents and if the preparation procedure seems to have been correct the stain may be good but the staining procedure was not correctly made Ensure that the proper staining method was followed Repeat with few more control slides paying attention to employing correct staining technique If no error is found in the preparation method or staining technique then prepare fresh staining solution s or reagents from a new batch of stains or reagents and perform quality control Report the unsatisfactory batch and discard the unsatisfactory solution s QUALITY CONTROL OF STORED STAINING REAGENTS Staining reagents may spoil with aging In addition the staining procedure may not have been performed correctly For these reasons check staining periodically in all labs Include a positive control smear as described above and prepared by these laboratories themselves in the routine series Perform the QC at least weekly and with each new batch of reagents Check the control smear first for properly stained AFB and record the result in the sputum smear register If the result is unsatisfactory sta
100. trate safe and correct collection of sputum Describe options for specimen collection handling and transport List features of a good sputum specimen Describe the requirements for a properly labeled specimen Transmission of TB bacilli Proper collection of sputum Laboratory arrangement Safety practices in the TB microscopy laboratory Safe disposal of infectious waste Chemical safety Suitable specimen containers The number and timing of specimen collection How to collect a specimen Specimen handling and referral Assessing specimen quality None None Module 4 Safety Precautions for TB Microscopy Page 1 of 13 Module 4 Safety Precautions for Tuberculosis Microscopy The most important factor in the prevention of laboratory acquired infection is good technique on the part of the individual worker Specialized equipment can support good laboratory practice but does NOT replace it Aerosols may be produced in the TB laboratory when handling leaking specimens opening sample containers and preparing smears When care and appropriate techniques are used handling sputum presents a minimal risk of acquiring infection to a technician For laboratory staff the greatest risk of infection involves sputum collection People with suspected TB may cough and in doing so spread TB bacilli in tiny droplets in the air which may infect others when they are inhaled Precautions must be taken to minimize this exposure The laboratory te
101. urfaces of lenses and prisms This can cause cloudiness of the view field and rusting of metal parts of the microscope To protect from fungus always keep the glass surface as clean as possible and free of dirt and fingerprints In very humid areas keeping the microscope inside a temperature controlled cabinet and using silica gel desiccant or anti mold strips may be useful Maintenance instructions Cover the instrument with the dust cover after every use Cover open tubes with the dust caps Remove dust and loose dirt from visible optical surfaces with a brush blower brush cotton bud optics cleaning tissue or a cotton cloth You may also use a cloth moistened with water to which you may add a mild detergent For resistant dirt use optics cleaning solution L 90 vol gasoline and 10 vol isopropanol Clean optical surfaces by gently wiping the objective lens in small circles starting in the middle and moving to the edges Never use petroleum benzene acetone or xylene to clean objective lenses TROUBLESHOOTING There are several conditions that can affect good functioning of the microscope Review these problems and their solutions The brightness contrast of the viewing field is poor Contrast Problem Solution FL Transmitted light slider is Always close slider when in fluorescence mode open BF Condenser is too low Raise the condenser to correct its position BF Condenser iris diaphr
102. ve slides are used at Yes No least once a week VI Supervisory site tasks Supply study forms Yes _ Not required Supply slide boxes Yes Not required _ Supply staining solutions Auramine Yes _ Not required _ at least once a month Completed forms and slides retrieved Yes _ for re checking VII Overall remarks foundation for innovative new diagnostics 71 avenue Louis Casal PO Box 93 CH 1216 Cointrin Geneva Tel 41 22 710 05 90 Fax 41 22 710 05 99 info finddiagnostics org www finddiagnostics org
103. with soap and water at least before and after performing any procedures Establish airflow in working areas that will direct potentially infectious particles away from personnel Air must be exhausted into a remote area An extraction fan can be useful to vent air from a smear preparation area with poor ventilation that is closed off due to extreme climatic conditions Do not rely on laboratory coats to protect you against infection with TB They are useful protection against strong chemicals staining reagents and accidental spills but they will not prevent TB infection Prepare smears near a spirit lamp flame Always follow safety procedures Module 4 Safety Precautions for TB Microscopy Page 3 of 13 Gloves Gloves do not provide any appreciable protection against airborne transmission of M tuberculosis Gloves are not required to prepare sputum smears and lack of their availability does NOT mean that sputum smears cannot be prepared Indeed wearing gloves can give technicians a false sense of safety and may result in contaminated gloves being used to handle or operate equipment that may otherwise not become contaminated e g microscope or telephone If gloves are used there should always be an adequate supply Reusing single use gloves is not advised Never wear gloves outside the laboratory Discard gloves at any interruption of smear preparation All gloves should be discarded in a foot operated closed lid waste receptacle c

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