Specimen Collection, Transportation and Storage in Medical Microbiology PDF

Specimen Collection,Transport and Storage Prof Olowo Samuel Dept of Medical Microbiology, College of Medicine, Ambrose Alli University, Ekpoma. Laboratory investigations Investigations carried out in; clinical diagnostic laboratories facility (hospital/clinic) based public health clinical research l...

Specimen Collection,Transport and Storage Prof Olowo Samuel Dept of Medical Microbiology, College of Medicine, Ambrose Alli University, Ekpoma. Laboratory investigations Investigations carried out in; clinical diagnostic laboratories facility (hospital/clinic) based public health clinical research laboratories non-clinical research laboratories Investigations in clinical diagnostic laboratories require quality assurance that involves; Process (describes work flow) Pre-analytical Analytical Post-analytical Procedure (actions carried out) Standard operating procedure for each process – document that specifies chronologic step-by-step actions needed Pre-analytical process patients preparation, specimen collection, handling, transportation, storage Analytical process tests conducted on the samples in the laboratory e.g. microscopy, staining, culture etc, personnel involved and equipment used Post-analytical process result interpretation, recording, reporting, dissemination of results, laboratory waste disposal etc Collection, transportation and storageof microbiological specimens the quality of the specimen submitted to the Clinical Microbiology laboratory determines the result generated if specimens of poor quality are submitted, the results generated by the laboratory will have little or no clinical utility. only specimens of good quality, i.e., those most likely to be useful clinically, are processed in the microbiology laboratory. You must understand the procedures for: patient preparation, specimen collection, processing and transport General rules for microbiological sample collection 1. Use standard precautions for collecting and handling all specimens 2. Whenever possible, collect all culture specimens prior to administration of any antimicrobial agents 3. Avoid contamination with indigenous flora 4. Swabs are convenient but inferior to tissue and fluid. Tissue and fluid are essential for fungal and mycobacterial culture 5. All specimens must be appropriately labeled patient identifiers. patient name, age/birth date, hospital number. requisition form should include patient name, hospital number, hospital service, date and time of collection, specimen type and tests requested requisition should accompany each different specimen type 6. All specimens must be delivered to the laboratory as soon as possible after collection. specimens for bacterial culture should be transported at room temperature. If transport is delayed the following specimens should be refrigerated: urine (within 30 min) stool (within 1 h) respiratory specimens except for suspected N. meningitidis specimens for viral culture must be transported to the laboratory immediately on ice. 7. Specimens should be in tightly sealed, leak proof containers and transported in sealable, leak-proof plastic bags. specimens for TB should be double bagged. specimens should not be externally contaminated. specimens grossly contaminated or compromised may be rejected. 8. If anaerobic culture is requested, proper anaerobic collection containers must be used Key issues in specimen collection Consider differential diagnoses Decide on test(s) to be conducted Decide on clinical samples to be collected to conduct these tests consultation between Microbiologist, Clinicians and Epidemiologist Transport medium Allows organisms (pathogens and contaminants) to survive Non-nutritive - does not allow organisms to proliferate For bacteria – i.e., Cary Blair For viruses - virus transport media (VTM) 1. Blood for smears Collection Capillary blood from finger prick make smear fix with methanol or other fixative Handling and transport Transport slides within 24 hours Do not refrigerate (can alter cell morphology) 2. Blood for cultures Collection Venous blood infants: 0.5 – 2 ml children: 2 – 5 ml adults: 5 – 10 ml Requires aseptic technique Collect within 10 minutes of fever if suspect bacterial endocarditis: 3 sets of blood culture Handling and Transport Collect into bottles with infusion broth change needle to inoculate the broth Transport upright with cushion prevents haemolysis Wrap tubes with absorbent cotton Transport at ambient temperature Store at 4oC if can’t reach laboratory in 24h 3. Serum Collection Venous blood in sterile test tube let clot for 30 minutes at ambient temperature glass better than plastic Handling Place at 4-8oC for clot retraction for at least 1-2 hours Centrifuge at 1 500 RPM for 5-10 min separates serum from the clot Transport 8 oC if transport lasts less than 10 days Freeze at -20oC if storage for weeks or months before processing and shipment to reference laboratory Avoid repeated freeze-thaw cycles destroys IgM To avoid haemolysis: do not freeze unseparated blood 4. Urine for microscopy & culture Collection Suprapubic aspiration (SPA) best method to avoid urine contamination invasive & therefore impracticable Straight single catheter technique next best method after SPA also invasive & impractical risk of introducing bacteria into bladder Clean-catch midstream voided urine simple & inexpensive not invasive risk of contamination by distal urethra flora not applicable in children, elderly and mentally challenged individual strict instruction to be followed to minimize contamination Catheter urine specimen use sterile technique collect urine from collection port using sterile needle & syringe maintain closed catheter drainage system while collecting urine DO NOT collect urine from urine bag preferably collect urine from a freshly inserted catheter (not old catheter) Handling urine should be collected in sterile leak proof container Transportation urine should be transported immediately to the laboratory for processing if a delay of >2 hour is envisaged, refrigerate or preserved during transport if delay of > 24 hour, use transport device with boric acid as preservative 5. Cerebrospinal fluid (CSF) Collection Lumbar puncture (4th/5th lumbar space) Sterile tubes Aseptic conditions Trained person Handling and transportation Bacteria preferably in trans-isolate medium, pre-warmed to 25-37°C before inoculation OR transport at ambient temperature (relevant pathogens do not survive at low temperatures) Viruses transport at 4-8oC (if up to 48hrs or -70oC for longer duration) 6. Stool samples Collection: Freshly passed stool samples avoid specimens from a bed pan Use sterile or clean container do not clean with disinfectant During an outbreak - collect from 10-20 patients Stool samples for viruses Timing within 48 hours of onset Sample amount 5-10 ml fresh stool from patients (and controls) Methods fresh stool unmixed with urine in clean, dry and sterile container Storage refrigerate at 4oC; do not freeze store at -15oC - for Ag detection,polymerase chain reaction (PCR) Transport 4oC (do not freeze); dry ice for (Ag detection and PCR) Stool samples for bacteria Timing during active phase Sample amount and size fresh sample and two swabs from patients, controls and carriers (if indicated) Method Cary-Blair medium For Ag detection/PCR – no transport medium Storage refrigerate at 4oC if testing within 48 hours, -70oC if longer; store at -15oC for Ag detection and PCR Transport 4oC (do not freeze); dry ice for Ag, PCR detection Stool samples for parasites Timing as soon as possible after onset Sample amount and size at least 3 x 5-10 ml fresh stool from patients and controls Method mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative unpreserved samples for Ag detection and PCR Storage refrigerate at 4oC; store at -15oC for Ag detection and PCR Transport 4oC (do not freeze); dry ice for antigen detection and PCR 7. Rectal swabs Advantage convenient adapted to small children, debilitated patients and other situations where voided stool sample not feasible Drawbacks no macroscopic assessment possible less material available not recommended for viruses 8. Throat swab (posterior pharyngeal swab) Hold tongue away with tongue depressor Locate areas of inflammation and exudate in posterior pharynx, tonsillar region of throat behind uvula Avoid swabbing soft palate; do not touch tongue Rub area back and forth with cotton or Dacron swab WHO/CDS/EPR/ARO/2006.1 9. Nasopharyngeal swab Tilt head backwards Insert flexible fine-shafted polyester swab into nostril and back to nasopharynx Leave in place a few seconds Withdraw slowly; rotating motion WHO/CDS/EPR/ARO/2006.1 10. Naso-pharyngeal aspirate Tilt head slightly backward Instill 1-1.5 ml of VTM /sterile normal saline into one nostril Use aspiration trap Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril WHO/CDS/EPR/ARO/2006.1 11. Wound specimen Wound biopsy superior to wound swab but invasive full thickness biopsy of wound put biopsy material in transport medium Wound discharge/abscess aspirate fluid/pus with sterile needle and syringe put in transport medium Wound swab use sterile cotton swab swab edge of wound put swab in transport medium problem of contamination by skin flora 12. Genital specimen Urethral swab massage the male penile shaft collect discharge from urethral meatus on swab put swab in transport medium High vaginal and endocervical swab use speculum to expose the vaginal/endocervix swab posterior fornix or endocervix process specimen immediately or put swab in transport medium 13. Sputum Collection Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container avoid saliva or postnasal discharge 1 ml minimum volume 14. Respiratory samples Handling and Transport All respiratory specimens except sputum are transported in appropriate media bacteria: Amie’s or Stuart’s transport medium viruses: viral transport medium (VTM) Transport as quickly as possible to the laboratory to reduce overgrowth by oral flora For transit periods up to 24 hours ambient temperature for bacteria 4-8°C for viruses 15. Post-mortem samples Collection Biopsy relevant tissues place in formalin for histopathology place in transport medium for microbiological testing place in sterile saline for isolation of viral pathogens Handling and transportation Fixed specimens can be transported at ambient temperatures transport specimens in transport media within 24h at ambient temperature transport specimens in sterile saline at 4-8oC within 48h 16. Virologic Investigations Transport Storage condition media Transport Pending test Purpose/ Lab investigation Throat swab VTM 2-8 0C -20 0C Isolation NPA/ swab VTM 2-8 0C -20 0C Isolation CSF No 2-8 0C -20 0C Isolation, serology Stool No 2-8 0C -20 0C Isolation Urine No 2-8 0C -20 0C Isolation Serum/ No 2-8 0C -20 0C Isolation, serology Specimen 2-8 0C Clotted blood Whole blood No 2-8 0C 2-8 0C Isolation, serology 17. Water for bacteriology Preparation Chlorinated water - add sodium thiosulphate (0.5ml of 10% solution or a small crystal) Tap/ pump remove attachments wipe, clean and flame outlet allow to flow (at least one minute) Water course or reservoir - collect from a depth of at least 20 cm Dug well - do not allow the bottle to touch the sides of the well Collection At least 200 ml of water sample from the source In sterile glass bottles OR autoclavable plastic bottles tight screw capped lid securely fitting stoppers/caps an overhanging rim Handling and transportation Test the water sample within 3 hours of collection keep at ambient temperature If delayed: pack sample on ice test refrigerated sample within 24 hours 18. Food samples Collect suspect food earliest Collect aseptically - sterile tools, containers Solid Food cut 100-200 grams from centre with sterile knife raw meat or poultry - refrigerate in a sterile plastic jar Liquids shake to mix, use sterile tube water used for cooking -- 1-5 litres Contact surfaces (utensils and/or equipment) for food processing moisten swab with sterile 0.1% peptone water or buffered distilled water; put the swab in an enrichment broth Handling and transportation As fast as possible Keep perishable food at 2-8oC Cool hot food rapidly - put containers under cold running water Pack samples to prevent spillage Contact the laboratory regarding method of transport and anticipated time of receipt Seek help from environmental/veterinary microbiologist Labeling specimens Labeling specimen container Patient’s name Clinical specimen Unique ID number (Research/Outbreak) Specimen type Date, time and place of collection Name/ initials of collector Labeling slides Label slides individually use glass marking pencil ensure markings don’t interfere with staining process Each slide should bear: patient name unique identification number date of collection Case/Laboratory investigation form Requesting physician or field epidemiologist fills form: Patient information age (or date of birth), sex, complete address Clinical information date of onset of symptoms, clinical and immunization history, risk factors or contact history where relevant, anti-microbial drugs taken prior to specimen collection Specimen detail information acute or convalescent specimen Date/time of collection Investigation required Detail of the sender Name and initials of person sending specimen Department/Address Signature Receiving laboratory records: Date and time when specimen was received Name and initials of the person receiving specimen Record of specimen quality Laboratory request form Biosafety: Protect the patient Use single use equipment Disinfect Work in a clean, dedicated area Protect yourself Use personal protective equipment disposable gloves laboratory coats / gown mask protective eyewear / face shields if procedure is likely to generate aerosols If no sharps container: collect sharps immediately to prevent needle-stick injury Have first aid kit readily accessible Do not reuse contaminated equipment Protect others & the environment Package samples appropriately for transport Decontaminate spills - 10% bleach after wiping the surface clean Disinfect working areas for future use - 1% household bleach daily Soak contaminated non-disposable equipment/material in 1% household bleach for 5 minutes wash in soapy water before re-use, sterilize if necessary Place waste in leak-proof biohazard bags - ensure safe final management of waste Protect cleaning/decontamination personnel with protective coat, thick rubber gloves Criteria for rejecting samples Mismatch of information on the label and the request form Inappropriate transport temperature Excessive delay in transportation Inappropriate transport medium specimen received in a fixative dry specimen sample with questionable relevance Insufficient quantity Leakage WHO reference materials Guidelines for the collection of clinical specimens during field investigation of outbreaks, WHO, 2000 The role of laboratories and blood banks in disaster situations, WHO publication, 2001 Sampling during avian influenza investigations (2006) IDSR guidelines for specimen collection (2003) Laboratory Needs for Emergency Situations (2003) Overview of Laboratory Structure and Operational Needs for the Iraqi Crisis (2003) Costing for sampling materials and diagnostic reagents for the Iraq crisis (2003) Thanks for listening.