MANUAL FOR LABORATORY TECHNICIANS

1 revised national tuberculosis control PROGRAMME (RNTCP) MANUAL FOR LABORATORY TECHNICIANSC entral TB Division, Directorate General of Health ServicesMinistry of Health and Family Welfare, Nirman Bhavan, New Delhi 110 011 revised national tuberculosis control PROGRAMME (RNTCP) MANUAL FOR LABORATORY TECHNICIANSF irst Printing: July 1997 Second Printing: September 1997 Third Printing: May 1999 Central TB Division, Directorate General of Health ServicesMinistry of Health and Family Welfare, Nirman Bhavan, New Delhi 110 011 CONTENTSA nnexuresICare of the microscope22 IIHow to use the microscope23 IIIC olour plates of AFB24 IVPrevention and consequences of false-positive26and false-negative sputum resultsVTroubleshooting guide for microscopy27 VIJob responsibilities of the Laboratory28 Technician (LT) in the revised NationalTuberculosis control Programme (RNTCP)VIIJob responsibilities of Senior Tuberculosis29 LABORATORY Supervisors (STLS) in the RevisedNational tuberculosis control Programme(RNTCP)

2 VIIIL aboratory Form for Sputum Examination30 IXTuberculosis LABORATORY Register31 Aims of sputum microscopy1 Advantages of sputum microscopy1 Layout of the microscopy laboratory1 When to collect sputum samples1 How to collect sputum samples2 How to prepare slides for examination6 How to examine slides under the microscope12 How to record and report results15 How to prepare stains and reagents17 How to ensure an adequate and continuous18supply of stains, reagents and other to dispose of contaminated20materials safelyHow to ensure quality of sputum microscopy2112345678910111211lllllMore reliable diagnostic tool than X-ray for thediagnosis of infectious TBlllllSimple to performlllllEasy to readlllllMinimal infrastructure required to set up aMicroscopy CentrelllllInexpensivelllllQuick resultslllllNecessary to monitor patient progress and todeclare the patient as "cured"Layout of the Microscopy LaboratoryA Microscopy LABORATORY should have areas to.

3 LReceive sputum sampleslPrepare and stain sputum smearslExamine slides under the microscopelTemporarily store contaminated materialslRecord results in the LABORATORY Form andLaboratory RegisterlStore boxes containing positive and negative slideslStore slides, reagents, forms and other to collect sputum samplesFor diagnosis of tuberculosis , 3 sputum examina-tions (SPOT MORNING SPOT) are performed. Forfollow-up of a case of tuberculosis , 2 sputum examina-tions (MORNING SPOT) are performed. Patientstaking different categories of treatment have their sputumexamined for follow-up at different times depending onthe categories of treatment and results of the first follow-up specimen (see Table 1).

4 The aims of sputum microscopy are patients with infectious the progress of tuberculosis patients who areon of sputum microscopyAims of sputum microscopy121212121212342 How to collect sputum samplesReceive the patient and LABORATORY FormCheck LABORATORY Form for completeness andaccuracyThe LABORATORY Technician (LT) can help patients byshowing genuine concern and patience. Emphasise thatdiagnostic facilities and treatment are free and thattuberculosis (TB) can be cured simply by taking regularand complete treatment as the LABORATORY Serial No. on the LaboratoryForm and sputum containerLaboratory Serial No.: When the patient comes for diag-nosis, all of his 3 sputum samples are given one Labora-tory Serial No.

5 When the same patient comes for sputumexamination at the end of 2 months, both follow-up samples are given a single new LABORATORY Serial the patient comes for the next follow-up sputumexamination, both his samples are given another newLaboratory Serial No. In brief, each patient is given oneCategory of treatmentSchedule of follow-up sputum examinationsSmear-positive Category IAt the end of 2, 4 and 6 months of treatmentSmear-positive Category IAt the end of 2, 3, 5 and 7 months of treatment(If sputum-positive at the end of Month 2)Smear-positive Category IIAt the end of 3, 5 and 8 months of treatmentSmear-positive Category IIAt the end of 3, 4, 6 and 9 months of treatment(If sputum-positive at the end of Month 3)Smear-negative Category I or Category IIIAt the end of 2 and 6 months of treatmentTable 1.

6 Schedule of sputum examinations53 Explain the difference between sputum and saliva,and the importance of bringing out sputum for examina-tion and make sure the patient understands. All sputumcontainers should be used only samplesGive the patient the labelled container, and bring him tothe nearby open space far away from other people, andthen instruct him by demonstrating with actual actions to:lInhale deeply 2 3 times;lCough out deeply from the chest;lOpen the container, bring it close to the mouthand bring the sputum out into it;lNot give saliva or nasal secretions;lClose the LABORATORY Serial No. for each set of sampleshe submits. The LABORATORY Serial No.

7 Begins with 1 on 1 January each year and increases by one with each pa-tient until 31 December of the same the LABORATORY Serial No. on the LaboratoryForm, and on the side of the sputum container, neveron the lid. This is because the lid from one container maybe placed on another container, causing incorrect label-ling of specimens. Write the LABORATORY Serial No. clearlyon the container using a permanent the sputum specimen from the patientGive the patient the sputum container with the LaboratorySerial No. written on the side of the container. Demon-strate to the patient how to open and close the containerand explain the importance of not rubbing off the numberwritten on the side of the good sputum container islllllDisposablelllllEasily burntlllllCleanlllllMade of clear thin plasticlllllWide-mouthedlllllLeak-proofl llllUnbreakablelllllProvided with tight-fitting lid3244 Before the patient leaves the LABORATORY , visuallyexamine the sputum sample for quality.

8 If the sample isonly saliva, ask the patient to cough again until a goodquality sample is obtained. A good quality sample mayrequire repetition of the procedure several the patient another container with the sameLaboratory Serial No. written on its side for an earlymorning specimen. Repeat the above instructions forbringing out sputum, adding that the patient shouldrinse his/her mouth with plain water before bringingup the early morning sputum specimen. This is to keepthe sample free of food particles. Food particles canappear like AFB under the microscope and can give afalse-positive of the sputumIf there is no microscopy centre easily accessible tothe patient, sputum can be collected in plastic sputumcontainers and transported to the nearest designatedmicroscopy centre.

9 For this purpose, a wooden box ofappropriate size with space to lodge sputum containerstightly can be made sputum should be:lcollected in a clean containerlkept in a cool place or in a refrigerator until transportedlprotected from excessive heat and direct sunlightlsent to the nearest designated microscopy centrepreferably by the next day and definitely within method of bringing out sputumcough324 Wooden box withsputum containers5 The Specimen Identification No. in all such cases isgiven only by health workers and others who are collect-ing sputum specimens and transporting the containers tothe microscopy centre for examination. However, allpatients including these patients, will have a LaboratorySerial upper portion of a LABORATORY Form for sputumexamination is reproduced below:Assess and record visual appearance of thesampleA good sputum sample is:lllllthick (semi-solid), coughed out deeply from the lungs;lllllpurulent (yellowish mucus);lllllsufficient in amount (at least 2 ml).

10 A poor quality sputum sample:lllllcontains only saliva (watery fluid) or nasal mucus;lllllis small in quantity (less than 2 ml).Make sure the sputum sample is of good quality formicroscopic examination. A good sample increases thechances of detecting portion of the LABORATORY Form where this infor-mation must be entered is reproduced on the next the visual appearance by writing M, B or S in theappropriate column. Make sure the LABORATORY Serial the form matches the LABORATORY Serial No. on the LABORATORY Serial No. on the slide with adiamond the specimen containers in serial order. Ensurethat the LABORATORY Serial Nos. on the sputum containersmatch the LABORATORY Serial Nos.