TISSUE COLLECTION. SCPA 603- Histopathological Techniques for Routine and Research

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1 TISSUE COLLECTION SCPA 603- Histopathological Techniques for Routine and Research Somphong Narkpinit, MD. Department of Pathobiology Faculty of Science, Mahidol University

2 Learning objectives At the end of the presentation, participants should understand the: Procedures, preparation, processing and transport of specimens

3 Successful laboratory investigations Advance planning Collection of adequate and appropriate specimens Sufficient documentation Biosafety and decontamination Correct packaging Rapid transport Choice of a laboratory that can accurately perform the tests Timely communication of results

4 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)

5 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)

6 Blood for cultures Collection Venous blood infants: 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

7 Blood for cultures Handling and Transport Collect into bottles with infusion broth change needle to inoculate the broth Transport upright with cushion prevents hemolysis Wrap tubes with absorbent cotton Travel at ambient temperature Store at 4 o C if can t reach laboratory in 24h

8 Serum Collection Venous blood in sterile test tube let clot for 30 minutes at ambient temperature glass better than plastic Handling Place at 4-8 o C for clot retraction for at least 1-2 hours Centrifuge at RPM for 5-10 min separates serum from the clot

9 Serum Transport 4-8 o C if transport lasts less than 10 days Freeze at -20 o C if storage for weeks or months before processing and shipment to reference laboratory Avoid repeated freeze-thaw cycles destroys IgM To avoid hemolysis: do not freeze unseparated blood

10 Cerebrospinal fluid (CSF) Collection Lumbar puncture Sterile tubes Aseptic conditions Trained person

11 CSF Handling and transportation OR Bacteria preferably in trans-isolate medium, pre-warmed to C before inoculation transport at ambient temperature (relevant pathogens do not survive at low temperatures) Viruses transport at 4-8 o C (if up to 48hrs or -70 o C for longer duration)

12 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 patients

13 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

14 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 4 o C; do not freeze store at -15 o C - for Ag detection,polymerase chain reaction (PCR) Transport 4 o C (do not freeze); dry ice for (Ag detection and PCR)

15 Stool samples for bacteria Timing during active phase Sample amount and size fresh sample and two swabs from patients, controls and carriers Method (if indicated) Cary-Blair medium For Ag detection/pcr no transport medium Storage refrigerate at 4 o C if testing within 48 hours, -70 o C if longer; store at -15 o C for Ag detection and PCR Transport 4 o C (do not freeze); dry ice for Ag, PCR detection

16 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 4 o C; store at -15 o C for Ag detection and PCR Transport 4 o C (do not freeze); dry ice for antigen detection and PCR

17 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

18 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

19 Naso-pharyngeal aspirate Tilt head slightly backward Instill 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

20 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

21 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

22 Post-mortem samples Collection Biopsy relevant tissues place in formalin for histopathology place in transport medium for microbiological testing place in sterile saline for prevent drying

23 Labeling specimens Patient s name Clinical specimen Unique ID number (Research/Outbreak) Specimen type Date, time and place of collection Name/ initials of collector

24 Case investigation form Receiving laboratory records: Date and time when specimen was received Name and initials of the person receiving specimen Record of specimen quality

25 Tissue collection Research Diagnosis Educated samplings

26 Tissue collection in research and diagnosis should be Organ good adequate fixation - histopathological technique : 10% formalin - immunohistochemistry : frozen fixation Identified normal and abnormal area Identified other associated abnormal findings

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37 Biosafety: protect the patient Use single use equipment Disinfect Work in a clean, dedicated area

38 Biosafety: 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 needlestick injury Have first aid kit readily accessible Do not reuse contaminated equipment

39 Thank you