Specimen Collection, Handling, Transport And Processing
Specimen Collection,Handling, Transport andProcessing1
Part 1:Specimen CollectionHandling and Transport2
Specimen Quality is ImportantThe results of tests, as they affect patientdiagnosis and treatment, are directlyrelated to the quality of the specimencollected and delivered to the ious/tuberculosis/Pages/tbtool.aspx3
Working with Healthcare Providers Laboratories must develop a good working relationshipwith health care providers collecting patient specimens Laboratories should have a reference manual forproviders that includes:– Specimen type and volume requirements– Specimen collection, labeling, storage and transportinstructions– Specimen rejection criteria Laboratories should provide specific feedback toindividual healthcare providers regarding problems withthe quality of specimens received and providerecommendations for improvement4
Specimen TypesI. Respiratory Sputum(expectorated,induced) Bronchoalveolarlavage (BAL) Bronchialwash/brush TranstrachealaspirateII. Non-respiratory Tissue Body fluids Blood Stool Gastric lavage UrineRefer to the CLSI M48-A document, Laboratory Detection andIdentification of Mycobacteria5
Specimen Collection, Handling, Transport and ProcessingRESPIRATORY (PULMONARY)SPECIMENS6
Sputum Recently discharged material from the bronchial tree, withminimal amounts of oral or nasal material Expectorated Sputum: Generated from a DEEP productive cough Induced Sputum: produced with hypertonic saline if patient isunable to produce sputum on their own Indications for sputum collection– To establish an initial diagnosis of TB– To monitor the infectiousness of the patient– To determine the effectiveness of treatment7
Sputum Quality Specimens are thick and containmucoid or mucopurulent material. Ideally, 3–5 ml in volume, althoughsmaller quantities are acceptable ifthe quality is satisfactory. Poor quality specimens are thin andwatery. Saliva and nasal secretionsare unacceptable. Laboratory requisition form shouldindicate when a specimen is inducedto avoid the specimen being labeledas “unacceptable” quality.Clinical and Laboratory Standards Institute. Laboratory detection and identification ofmycobacteria; approved guideline. CLSI Document M48-A. Wayne, PA: CLSI; /29 specimen condition transport.doc8
Sputum atery(acceptable ifinduced)Salivary9
Indications for Sputum Collection Initial diagnosis of TB: Collect a series of three sputum specimens, 8-24 hoursapart, at least one of which is an early morning specimen Optimally, sputum should be collected before the initiationof drug therapy For release from home isolation: If patient is smear positive and on treatment: Collectsputum until 3 specimens are negative. Monitoring of therapy: Obtain sputum specimens forculture at least monthly until cultures convert tonegativeCenters for Disease Control and Prevention. Guidelines for Preventing the Transmission of Mycobacteriumtuberculosis in Health-Care Settings, MMWR 2005:54, RR-1710
Specimen Collection: All aerosol producingprocedures pose a risk of exposureWhether collecting specimen viasputum collection or bronchoscopy, ifa patient is suspected or confirmedof have tuberculosis, airborneprecaution must be used.11
Specimen Collection Suspect or confirmed TB patients shouldbe in a negative pressure room Specimen collection is an aerosolgenerating procedure, anyone in the roomduring specimen collection must wear aparticulate respirator type N-95 and bepart of the respirator protection plan All mycobacteria specimens are collectedinto a sealed leak proof container12
Storage and Transport of SputumSpecimens Collection sites should refrigerate samples thatcannot be transported immediately to reducegrowth of contaminating organisms Specimens should be delivered to the laboratoryas soon as possible, within 24 hours of collectionis optimal (avoid batching) Laboratories may include a cold pack withspecimen transport materials13
Pulmonary Specimens Other Than Sputum:Collection ransportBronchoalveolarlavage (BAL)Collect washingor aspirate insputum trapMinimum volumeis 3 ml50-ml conical tube orother sterile containerBronch brush Place the brushin a sterile, leakproof containerwith up to 5 mlof sterile salineTransport as soon aspossible at roomtemperatureIf transport is delayedmore than 1 hour,refrigerate specimen. The doubling time for other common respiratory flora is 15 to 20 minutes The doubling time for M. tuberculosis is 12 to 24 hours14
Specimen Collection, Handling, Transport and ProcessingNON-RESPIRATORY(EXTRAPULMONARY) SPECIMENS15
Extrapulmonary Specimens The laboratory should expect to receive a varietyof extrapulmonary specimens which may bedivided into two groups– specimens from non-sterile body sites– specimens from normally sterile body sites Should be collected in a sterile leak-proofcontainer Should be transported as soon as possible Swabs are generally not acceptable16
Extrapulmonary SpecimenCollection GuidanceSpecimenfrom NonSterile BodySitesRecommendedCollection TimeVolumeRequirementsGastricAspirateEarly morningbefore patienteats and whilestill in edfor Isolation ofMTBC?5–10 ml isOne specimenoptimal;per day on threemaximumconsecutive daysvolume is 15 mlRoomtemperature; ifdelayed 1hour, neutralizewith 100 mgsodiumcarbonateYesFirst morningspecimen (voidmidstream)10–15 mlminimum;prefer up to 40mlOne specimenper day on threeconsecutive daysIf delayed 1Yeshour, refrigerateNorecommendationMinimumvolume is 1gramNorecommendationRefrigerate ifdelayed 1hour, do notfreezeMainly fordiagnosis ofdisseminatedMAC disease inpatients with17AIDS
Extrapulmonary SpecimenCollection GuidanceSpecimen fromNormally Sterile BodySitesVolume RequirementsTransportRecommended forIsolation of MTBC?Cerebral Spinal Fluid10 ml is optimal;minimum volume is 2-3mlAs soon as possible atroom temperature; do notrefrigerateUsually paucibacillary;culture may have limitedsensitivityOther Body Fluids(pleural, peritoneal,pericardial, synovial)10-15 ml is optimal;If delayed, refrigerateminimum volume is 10 mlTissues or Lymph NodesAs much as possible;add 2-3 ml sterile salineAs soon as possible atYesroom temperature (noformalin, preservatives, orfixatives)Blood10ml preferred, minimum5 ml. Collect in SPS orheparin tube, no EDTAAt room temperature, donot refrigerate or freezeYesMainly for diagnosis ofdisseminated MACdisease in patients withAIDS18
Specimen Collection, Handling, Transport and ProcessingSUBOPTIMAL AND UNACCEPTABLESPECIMENS19
Suboptimal and UnacceptableSpecimens Processing of suboptimal or poor qualityspecimens is a burden on both financial andpersonnel resources Results generated from processing inappropriatespecimens may not be reliable Each laboratory must develop its own specimenrejection criteria and make these criteria readilyaccessible to providers Clinicians should be notified when a specimen isrejected and the reason for rejection should beprovided Specimens collected by invasive proceduresshould not be rejected20
Possible Rejection Criteria (1) Labeling of specimen does not match identifierson requisition form Insufficient volume Dried swabs– Swabs in general are not optimal Provide limited material Hydrophobicity of mycobacterial cell envelopeinhibits transfer to media Pooled sputum or urine Sputum left at room temperature for 24 hours21
Possible Rejection Criteria (2) Broken specimen containers or leakingspecimens Excessive delay between specimen collectionand receipt in the laboratory Blood specimens collected in EDTA might berejected for culture as these inhibit growth ofMTB Tissue or abscess material in formalin Gastric lavage fluid if pH not adjusted within 1hour of collection22
Specimen Collection, Handling, Transport, and ProcessingSPECIMEN TRANSPORT: REFERRAL OFSPECIMENS WITHIN A LABORATORYNETWORK23
Transport of Biological Substances(Category B)Basic triple packaging system (i) a leak-proof primary receptacle(s); (ii) a leak-proof secondary packaging containingsufficient additional absorbent material shall beused to absorb all fluid in case of breakage For cold transportation conditions, ice or dry ice shall be placedoutside the secondary receptacle. Wet ice shall be placed in a leakproof container. (iii) an outer packaging of adequate strength forits capacity, mass and intended use.24
Transport of Biological Substances(Category B)Biological Substance,Category B25
Sputum Collection Kit Insulated mailer with address label UN3373 marking and propershipping name “BiologicalSubstance, Category B” Sterile plastic conical tube withlabel Sealable biohazard specimentransport Cool pack Absorbent padAn example from the WisconsinState Laboratory of Hygiene Instruction sheet26
Transport of Biological Substances Transport of patient specimens is regulated byboth the Department of Transportation (DOT)and by International Air Transport Association(IATA) rules. Laboratories must have personnel trained in andfamiliar with these regulations For details regarding these regulations, pleasesee the information provided in the next slide.27
Packing and Shipping Guidance ASM website-Guidance: Packing and Shipping InfectiousSubstances DOT guidance: Transporting Infectious Substances Safely More DOT Guidance: Infectious substances guide IATA website: FAQs Infectious Substances FedEx Guidance: Clinical Samples, Biological SubstancesCategory B(UN 3373) and Environmental Test Samples UPS Guidance: Packing Hazardous Materials28
Specimen Collection, Handling, Transport and ProcessingADDITIONAL INFORMATION29
Instructions for Sputum Collection Healthcare providers should educate patients on proper specimenproduction and collection Patients should also be informed of the possible infectious nature ofhis or her secretions Specimens should be collected in appropriate tubes that are sterile,clear, plastic, and leak-proof (50 ml screw capped centrifuge tubesthat can withstand 3000 x g are preferred) Proper labeling protocols should be put in place by the laboratories Work with TB program to provide instructions for submitters30
Examples for Instructions forSputum CollectionPennsylvaniaDepartment ofHealth31
Part 2:Specimen Processing32
Principles of Specimen ProcessingRespiratory specimens (and other specimens from nonsterile sites) require digestion, decontamination, andconcentration: Digestion: Mucolytic agent used to liquefy sputumspecimens to release AFB and expose normal flora fordecontamination Decontamination: Toxic agent used to kill rapidly growingnormal flora that would otherwise overgrow slow growingmycobacteria Centrifugation: Used to sediment bacteria followingdigestion/decontamination33
Effect of Processing Procedure Reagents used for digestion and decontamination, tosome extent, are toxic to mycobacteria Procedures must be precisely followed–– Time in contact with digestion/decontamination reagents iscriticalCentrifugation procedures also affect mycobacteria recoveryrates and must be carefully followedCulture positivity rates and contamination rates shouldbe monitored to evaluate performance of processingmethods34
Safety Many of the steps within the specimen processingprocedure are aerosol-generatingThe following activities should be performed in abiological safety cabinet: Digestion and decontaminationPreparation of concentrated smearInoculation of culture mediaDo not disrupt airflow of the cabinetAvoid excess clutter inside the BSCFor more information on the biosafety practices in the Mycobacteriologylaboratory, please review the module on biosafety within this series35
Aseptic Techniques: Getting Started Disinfect BSC, centrifuge, and tabletop with tuberculocidaldisinfectant (repeat after specimen and culture workupcomplete) Perform all work on absorbent pad soaked with disinfectant toabsorb any droplets that may inadvertently occur When possible, leave an empty space in the rack betweeneach specimen tube Work in sets equivalent to one centrifuge load (e.g., 8specimens at a time)36
Aseptic Techniques: Processing Use a fresh individual, disposable, sterile pipette at every step toavoid transfer of bacilli To avoid droplet aerosol cross-contamination, open and removecaps from tubes one at a time Add diluent to centrifuge tubes from individual tubes without the lipof the tube touching or creating an aerosol (do not use commoncontainers or carboys) Use aerosol-proof sealed centrifuge cups Discard supernatant from decontaminated specimens into splashproof container with disinfectant. Autoclave the discard containerdaily37
Digestion & Decontamination MethodsSeveral methods are available for digestion anddecontamination of clinical specimens: N-acetyl-L-cysteine-sodium hydroxide (NALCNaOH) Commercially available: Alpha-Tec NAC-PAC , BDMycoPrep Oxalic acidCetylpyridinium chloride (CPC)-sodium chlorideNaOH method (Petroff’s method)Zephiran-trisodium phosphate (Z-TSP)38
NALC-NaOH Method: Principle Most common and preferred method Rapid and relatively effective in reducing the number ofcontaminants Addition of the mucolytic agent 2% NALC allowseffective decontamination with 1% NaOH (less harsh onmycobacteria) (final concentrations) Sodium citrate also included in digestion mixture to bindheavy metal ions in specimen that could inactivate Nacetyl-L-cysteine39
NALC-NaOH Method: Reagents NaOH and sodium citrate can be prepared andcombined in advanceNALC should be prepared and added fresh dailyOnce NALC added, solution should be used within 24hoursCLSI M-48AKent and KubicaVolume ofdigestantneeded4% NaOH2.9% sodiumcitratedehydrateAdd NALC(fresh)50 ml25 ml25 ml0.25 g100 ml50 ml50 ml0.50 g500 ml250 ml250 ml2.50 g40
NALC-NaOH Method: Procedure for Sputum1.Add equal volume of NALC-NaOH solution to 5-10 ml of sputum in50 ml plastic screw cap centrifuge tube2.Cap tube tightly. Invert the tube so that the NALC-NaOH solutioncontacts all inside surfaces of the tube and cap and then mix thecontents for approximately 5-20 seconds with a Vortex mixer.3.Allow mixture to stand for 15 minutes at room temperature withoccasional gentle shaking by hand.4.Add sterile distilled water or sterile pH 6.8 phosphate buffer to the50 ml mark on tube. Securely cap tube and mix by inversion5.Centrifuge the tubes for 15 min at 3,000 x g using aerosol-proofsealed centrifuge cups41
NALC-NaOH Method: Procedure for Sputum After centrifugation, open centrifuge cups in BSC, slowly poor offsupernatant into splash-proof discard container containingdisinfectant––Take care to not disturb or pour off sediment (pellet)Take care to avoid aerosol production and to prevent contamination ofthe lip of the tube Wipe the lip of the tube with gauze soaked with disinfectant Resuspend sediment in 1-2 ml of saline solution or phosphate buffer Mix gently and proceed to culture inoculation and smear preparation42
NaOH Concentration is Key Recommended final concentration is 1.0% NaOH Concentration of NaOH can be adjusted based on contaminationrate in individual laboratories NaOH at a FINAL concentration of 2.0% can be lethal tomycobacteria (may see decrease culture sensitivity in smearnegative specimens)Amount ofNaOH in 100 mlwater% NaOH% NaOH whenadded to equalvolume NacitrateFinalconcentration ofNaOH whenadded 4.0%2.0%43CLSI M-48A
Timing is CRITICAL 15 MINUTES– Time in contact with NALC-NaOH is critical since the highpH rapidly kills microorganisms in the specimen includingmycobacteria Over processing results in reduced recovery ofmycobacteria The importance of timing must be considered whendeciding how many specimens can be processed inone run44
Specimen Processing QC A Negative Processing Control (10 ml sterilewater or buffer) should be included with eachbatch of specimens processed Negative control is put through entire specimenprocessing procedure and inoculated to media Determines if contamination is introduced duringprocessing or culture handlingAssures that isolates are coming from patients andnot from any other source45
Definition of Cross ContaminationCross contamination: the transfer of M.tuberculosis complex bacilli (or othermycobacteria) from one specimen toanother specimen that does not containviable bacilli, causing a false positive result.– The phenomenon of misdiagnosis of tuberculosis dueto cross contamination has been widely reported andhas significant clinical and therapeutic impact on thepatient.46
To reduce the possibility of CrossContamination: Use daily aliquots of processing reagents and buffers.Any leftover should be discarded. Never use common beakers or flasks when processing. Keep the specimen tubes tightly closed and clean theoutside of the tube prior to vortexing or shaking. Pour decontamination reagents or buffers slowly on theside of the tube without causing any splashing. Do nottouch the container of reagents to the lip of the tube atany time during addition.47
To reduce the possibility of CrossContamination: Open the specimen tubes very gently to avoid aerosolgeneration. When adding reagents to the tube, open one tube at atime. Do not keep all the tubes open at the same time. Do not place tubes too close to each other in the rack Change gloves often Avoid manipulation of PT specimens Disinfect biological safety cabinet work surfacesroutinely.48
Specimen Processing Proficiency Digestion, decontamination, and conentrationprocedures should only be performed byt rrainedlaboratory staff. Mycobacteriology laboratories should participate in anapproved proficiency testing program. Proficiency in culture and identification of MTV may bemaintained by digestion and culture of 15-20 specimensper week.49
Specimen Collection, Handling, Transport and ProcessingSPECIMENS FROM CYSTIC FIBROSISPATIENTS AND EXTRAPULMONARY SITES50
Cystic Fibrosis (CF) Patients Specimens from CF patients are often heavilycontaminated with Pseudomonas aeruginosa. If it is known or discovered specimen is from apatient with CF or notable media contaminationyou can process concentrated sediment usingonly the 5% oxalic acid method.51
Oxalic Acid Processing Method for Specimensfrom CF Patients Add an equal amount of 5% oxalic acid to:- 5-10 ml of primary respiratory specimen or- NALC-NaOH processed concentrated sediment Vortex specimen and allow to incubate at room temperature for 30minutes, mixing every 10 minutes Neutralize with buffer solution Concentrate specimen by centrifugation for 15 minutes at 3000 x g Decant supernatant into splash-proof container and resuspend pelletwith buffer solution Inoculate media52
Processing Gastric Lavage and Urine Centrifuge for 30 minutes at 3000 x g Discard supernatant carefully into splash-proofcontainer Re-suspend pellet in sterile distilled water Process suspension as for sputum (NALCNaOH)53
Processing Aseptically Collected Fluids Cerebral spinal fluid (CSF), synovial,pleural, peritoneal, pericardial No decontamination required Concentrate specimen to maximize theyield of mycobacteria Inoculate directly to culture media54
Processing Tissue Specimens Lymph node, lung tissue, biopsies Tissue submitted in formalin is unsatisfactory for culture No decontamination required Process tissue specimens using a sterile tissue grinder,or mortar and pestle Inoculate directly to culture media55
Processing Blood or Bone Marrow Aspirates Specimens inoculated directly into MYCO/F LYTICbottles or BacT/ALERT MB blood medium Direct inoculation of
Specimen Collection Suspect or confirmed TB patients should be in a negative pressure room Specimen collection is an aerosol generating procedure, anyone in the room during specimen collection must wear a particulate respirator type N-95 and be part of the respirator protection plan All mycobacteria specimens are collected
Specimen Collection Container CytoLyt Solution Biohazard Safety Bag Specimen Collection: 1. Label specimen container with 2 patient identifiers 2. Collect specimen on brush. 3. Cut off brush end and immediately submerge brush in CytoLyt to avoid air-drying effects Specimen Handling: Room temperature if specimen is delivered upon .
Specimen Collection Manual Page 4 of 35 HOW TO SEND A SPECIMEN TO LBM: - Use LBM requisition. - Place specimen in proper fixative. - Label specimen bottle with two patient's identifiers, i.e., name and date of birth. Also include specimen source, physician name & collection date. - Call LBM Client Services courier for pick up at (562) 989-5858 If you are already on the LBM courier's daily .
4 HOW TO SEND A SPECIMEN TO TOPA: -Use TOPA requisition.-Place specimen in proper fixative.-Label specimen bottle with two patient identifiers, i.e., name and date of birth.Also include specimen source, physician name & collection date. -Call TOPA courier for pick up at (805) 373-8582.If you are already on the TOPA courier's daily route, place specimen in your designated pick-up
Specimen Collection, Handling and Transport Page 5 of 6 1. Room Temp Requirements: If your specimen does not have a specific storage requirement and will be stored at room temp before Courier pickup, please place in a sealed orange/red
specimen cannot be processed within one to two hours of collection, they must be refrigerated. Additionally the specimen should ideally reach the laboratory within 24 hours of collection and be kept cold during transport. It is important that the submitter know to avoid batch shipment. Don't let them collect all three
The primary responsibility of the specimen collector is to collect urine samples while following universal . Cary Blair Stool Kit Genprobe Aptima Swab Genprobe Aptima Urine Green Top Tube 4. Specimen Type Requirements - Illustrations . Specimen Collection and Handling section of this directory.
Cytology Specimen Collection. For additional assistance with test ordering, . Quest Diagnostics performs testing on a wide range of sample types. Properly collecting and preparing . Specimen Collection Requirements. Refer to the digital tools explained below for expanded . instructions on patient preparation and laboratory specimen .
Scrum Development Team A self-organizing, self-managed cross-functional team responsible for delivering commitments from the Product Backlog. User Stories Describe what the end product and its components should accomplish at the end of development. A product will usually have multiple user stories. Product Backlog A list of features or technical tasks which the team maintains and which, at a .
