Principle of diagnostic methods collection storage and transport of specimens

Specimen collection, storage, and transport methods are described in detail as helpful for the students of medicine, laboratory medicine, and microbiology. The presentation is specifically focusing only on microbiology points of view while collecting specimens for laboratory investigations and diagnostic purposes.

1. Principle of Diagnostic Methods : Collection, Storage and Transport of Specimens UNDER GARADUATE STUDENT’S PRACTICAL ON BY GUNJAL PN ASSIST. PROF. DEPT OF MICROBIOLOGY DVVPF’S MEDICAL COLLEGE & HOSPITAL AHMENDAGAR 2/10/2021 Department of Microbiology 1

2. Competencies Following are the competencies for this practical class : MI 8.9 – Discuss the appropriate methods of collection of samples in performance of laboratory tests in detection of microbial agents causing infectious diseases. MI 8.10- Demonstrate appropriate methods of collection of samples in the performance of laboratory tests in detection of microbial agents causing infectious diseases. MI 8.11 – Demonstrate respect for patient samples sent to laboratory for performance of laboratory tests in the detection of microbial agents causing infectious diseases. MI 8.12 – Discuss confidentiality pertaining to patient identity in laboratory results. 2/10/2021 Department of Microbiology 2

3. Learning Objectives At the end of the session, the students will be able to understand : Site and type of specimens to be collected for laboratory diagnosis of microbiological investigations. Procedures, preparation, processing and transport of specimens. Procedures, preparation, processing for specimen storage for laboratory diagnosis. 2/10/2021 Dept of Microbiology 3

4. Introduction Specimen collection and transportation are critical considerations. Because any results the laboratory generates is limited by the quality of the specimen and its condition on arrival in the laboratory. Specimens should be obtained to minimize the possibility of introducing contaminating microorganisms that are not involved in the infectious process. 2/10/2021 Department of Microbiology 4

5. 2/10/2021 Department of Microbiology 5 General guidelines for specimen collection Advance planning- Discussion between with microbiologist, clinician, epidemiologist. Collection of Correct, 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 .

6. An Ideal Request form Name : Age : Sex : IP / OP No : Time & Date : Ward: Urgent / Routine : Nature of specimen : Investigation needed : Doctor/Staff Contact No : 1234567 2/10/2021 Department of Microbiology 6

7. Use sterile single use equipment. Disinfect. Work in a clean, dedicated area. 2/10/2021 Department of Microbiology 7 Biosafety: protect the patient

8. 2/10/2021 Department of Microbiology 8 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. Biosafety: protect yourself

9. 2/10/2021 Department of Microbiology 9 Biosafety: 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.

10. 2/10/2021 Department of Microbiology 10 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 ).

11. Specimen transport Within 2 hours of collection Containers should be leak-proof Separate section for paperwork Special preservatives or holding media Biohazard label 2/10/2021 Department of Microbiology 11

12. Triple packaging system 2/10/2021 Department of Microbiology 12 Triple packaging provides three layers of containment to protect the substances being shipped. These layers are primary, secondary, and outer containers. The following diagram shows the basic concept of triple packages.

13. 2/10/2021 Department of Microbiology 13 Primary Container Secondary Container Outer Container Triple packaging system

14. Category A packaging requirements 2/10/2021 Department of Microbiology 14

15. Criteria for rejection of specimens Several criteria can be considered by a laboratory on the basis of which the processing of a specimen may not be done by the laboratory. Such a decision must be made in light of the specific requested investigation . Laboratory investigations of a sample are a waste of time and resources if following criteria are not fulfilled : 2/10/2021 Department of Microbiology 15

16. Missing or inadequate identification  Insufficient quantity. Specimen collected in an inappropriate container  Contamination suspected. Inappropriate transport or storage. 2/10/2021 Department of Microbiology 16 Criteria for rejection of specimens

17. Containers and swab for the collection of specimens For Faeces: Universal container. Spoon attached to the inside of the screw cap. For Urine: Universal container for small quantities. For larger quantities 250 ml wide mouthed screw-capped bottles are convenient. 2/10/2021 Department of Microbiology 17

18. For sputum : Universal container should not be used. Squat ,wide-mouthed disposable containers should be used. 2/10/2021 Department of Microbiology 18 Containers and swab for the collection of specimens

19. For Blood: Without anticoagulant for serological examination . With EDTA for parasitological examination . Blood culture bottle: This must be at least large enough to hold 50ml of liquid medium ,with which it is issued from laboratory, plus 5-10ml of patient’s blood 2/10/2021 Department of Microbiology 19 Containers and swab for the collection of specimens

20. For serous fluids Universal container Addition of 0.3ml of 20% solution sodium citrate to the container prior to autoclaving (with the cap fitted) is recommended for collection of fluids that may coagulate on standing. This avoids difficulty in performing cell counts or centrifuging procedure with such fluids. 2/10/2021 Department of Microbiology 20

21. Swabs Swabs suitable for taking Specimens of exudates from the throat, nostril , ear , skin, wounds and other accessible lesions consist of a sterile absorbent material, usually cotton-wool or synthetic fiber, mounted on a thin wire of stick. Swabs for special purpose: Baby swabs Pernasal swabs Post-nasal swabs Laryngeal swabs High vaginal and cervical swabs Serum coated cotton wool swab 2/10/2021 Department of Microbiology 21

22. Containers of anaerobic specimens : Syringe and needle for aspiration: Tube or vial contains semi-solid holding medium an atmosphere of 5% CO 2 ,a reducing agent, tube used for putting up the swab. Readymade swabs in a plastic tube and containing either Cary-Blair , Amies transporter prereduced (PRAs) medium. Plastic pouch or Bio-bag (transparent) containing a CO 2 generating system, palladium catalyst and an anaerobic indicator can also be used. 2/10/2021 Department of Microbiology 22

23. EYE Various specimens collected are: A . specimens: 1.Conjunctival: Container : Aerobic swab moistened with Stuart’s or Amie’s medium. 2/10/2021 Department of Microbiology 23

24. Collection: Obtained from superior and inferior tarsal conjunctiva. Specimen of both eyes with separate swabs by rolling swab over each conjunctiva. If a viral culture is requested ; a second specimen is collected For Chlamydia culture swabs are taken with a dry calcium alginate swab 2/10/2021 Department of Microbiology 24

25. Transport : Within 24hrs/RT. For viral culture place in viral transport media and deliver promptly to laboratory or refrigerated for a short time and then transport on wet ice. For Chlamydia place in 2-Sp transport medium. 2/10/2021 Department of Microbiology 25 EYE

26. 2. Corneal scrapings Container : Bedside inoculation of BA, CA, SDA, 7H10, Thio. Patient preparation: Clinician should instil local anesthetic before collection. Collection : By using heat sterilized platinum spatula or calcium alginate tipped swab dipped in sterile trypticase soya broth. Transport : Immediately/RT 2/10/2021 Department of Microbiology 26

27. Anterior chamber and vitreous cultures Collection: Aspiration is carried out with a tuberculin syringe Fitted with a 25-27 gauge needle for the aqueous. 20-21 gauge needle for vitreous aspiration. Transport : Immediately/RT 2/10/2021 Department of Microbiology 27

28. EAR 1. Inner ear: Container : • Sterile , screw-cap tube or anaerobic transporter. Patient preparation: Clean ear canal with mild soap solution before puncture of the ear drum. Collection : Aspirate material behind drum with syringe if ear drum is intact; use swab to collect material from ruptured eardrum. Transport : Immediately/RT 2/10/2021 Department of Microbiology 28

29. 2. Outer ear: Container : Aerobic swab moistened with Stuart's or Amie’s medium  Patient preparation: Wipe away crust with sterile saline. Collection : Firmly rotate swab in outer canal. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 29

30. Respiratory tract(RT): Collection of specimen in the case of RTI poses a number of problems because , there is enormous commensal flora that colonizes this tract. Therefore , the specimen collection is very crucial and specially in case of viral infections of RT. One has to avoid contamination of the specimens. 2/10/2021 Department of Microbiology 30

31. RT is broadly divided into : A . Upper RT: Container : Swab moistened with Stuart’s or Amie’s medium. Collection : 1.Oral swab: Remove the oral secretions or debris from the surface of lesion with swab and discard. Using 2nd swab ,vigorously specimen the lesion avoiding any areas of normal tissue 2 . Nasal swab: Use swab moistened with sterile saline . Insert approx. 2cm into nares. Rotate swab against nasal mucosa 2/10/2021 Department of Microbiology 31 Respiratory tract(RT):

32. 3. Nasopharyngeal: A . Swabs: To collect nasopharyngeal cells, all mucus is removed. Small flexible nasopharyngeal swab is inserted along the nasal septum to the posterior pharynx. Rotate slowly for 5 sec. against the mucosa several times B . Aspirate : Is collected with a plastic tube attached to 10 ml syringe or suction catheter. C . Washings: Is obtained with a rubber suction bulb by instilling and withdrawing 3-7 ml of sterile buffer saline. 2/10/2021 Department of Microbiology 32

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34. Laryngeal swab Before use the swab is moistened with sterile D/W. Patient is made sit and holding the tongue fully protruded W ith help of a piece of gauge, pass the swab back through the mouth wire mid-line and downwards over the epiglottis into larynx where it should induce reflex coughing that will expel sputum onto swab. Withdraw the swab and replace it in its tube for delivery to the laboratory. 2/10/2021 Department of Microbiology 34

35. Throat swab Collection: Depress the tongue with a tongue depressor. Introduce the swab between the tonsillar pillars and behind the uvula without touching the lateral walls of the buccal cavity. Swab back and forth across the posterior pharynx. Any exudates or membrane should be taken for specimen. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 35

36. B. Lower RT Container: Sterile screw-top container. Collection : 1.Sputum: Patient preparation: Ask patient to brush teeth and then rinse or gargle with water before collection. Collected early in the morning before eating. Make collection in a disposable wide mouthed screw-capped sterile plastic container of about 100ml capacity. Instruct to wait until he/she feels material coughed into his/her throat. Then work it forward into mouth and spit it directly into container. Should be collected before starting antimicrobial chemotherapy. 2/10/2021 Department of Microbiology 36

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38. Obtained by inserting a small plastic catheter into the trachea via a needle previously inserted through the skin and cricothyroid membrane. This technique is rarely used any more 2/10/2021 Department of Microbiology 38 2. Transtracheal aspiration(TTA):

39. 3.Bronchioalveolar lavage (BAL): 30-50 ml of physiological saliva is injected through a fiberoptic bronchoscope . The saliva is then aspirated. 4.Gastric lavage: In the morning before the patient has taken anything but after a bout of coughing and swallowing , aspirate the fasting stomach contents with a Ryle’s tube. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 39

40. BODY FLUIDS 1.Cerebrospinal fluid: Container : Sterile screw-cap tube. Patient preparation: Disinfect skin before aspirating specimen. Collection: Lumbar puncture to collect the CSF for examination to be collected by Physician trained in procedure with aseptic precautions to prevent introduction of Infection . The trained physician will collect only 3-5 ml into a labelled sterile container. The fluid to be collected at the rate of 4-5 drops per second. 2/10/2021 Department of Microbiology 40

41. The best site for puncture is inter space between 3 and 4 lumbar vertebrae. The Physician should wear sterile gloves and conduct the procedure with sterile precautions. The site of procedure should be disinfected and sterile air and water tight (occlusive) dressing applied to the puncture site after the procedure 2/10/2021 Department of Microbiology 41

42. Transportation to Laboratory: The collected specimen of CSF to be dispatched promptly to Laboratory without any delay. Delay may cause death of delicate pathogens, e.g. Meningococci and disintegrate leukocytes. Preservation of CSF: It is important when there is delay in transportation of specimens to Laboratory do not keep in Refrigerator, which tends to kill H. Influenzae. If delay is anticipated leave specimen at Room Temperature. 2/10/2021 Department of Microbiology 42

43. 2.Pleural/Peritoneal/Pericardial/ Synovial fluid: Container : Sterile screw-cap tube or anaerobic transporter  Patient preparation: Disinfect skin before aspirating with 2% iodine tincture. Collection : Obtained via percutaneous needle aspiration or surgery. Transport : Immediately/RT. 2/10/2021 Department of Microbiology 43

44. BLOOD Container : Blood culture media set(aerobic and anaerobic bottle) or vacutainer tube with SPS ( sodium polyanethol sulfonate) prevents blood from clotting for 2-4 hrs. Patient preparation: Disinfect venipuncture site with 70% alcohol and disinfectant such as betadine. 2/10/2021 Department of Microbiology 44

45. Collection: Select the vein from which blood is to be drawn. Disinfect the venipuncture site. Allow it to dry. With precautions to avoid touching and recontaminating the venipuncture site. Take the specimen of blood and put it immediately through the hole in the cap of bottle . Volume of blood: In adult 5-10ml In children 1-5ml Transport: Within 2hrs/RT 2/10/2021 Department of Microbiology 45

46. Gastrointestinal tract(GIT ) 1.Stool : Container : Clean leak-proof container . Do not clean with disinfectants. Collection : Collect sample during active phase. Pass stool directly into a sterile, wide-mouth, leak proof container with a tight fitting lid. For parasitological investigation mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative Transport : Within 24hrs/4 ° C. If delay is unavoidable and particularly when the weather is warm collect the specimens in a container holding 6 ml buffered glycerol saline transport medium. 2/10/2021 Department of Microbiology 46

47. Transport media for stool specimens Cary-Blair - All enteric organisms. Stuart - All enteric organisms. Amies - All enteric organisms. Buffered All enteric organisms except Vibrios & glycerol Campylobacter. saline - Alkaline Vibrios peptone water - V-R fluid - Vibrios 2/10/2021 Department of Microbiology 47

48. 2.Rectal swab: Container : Swab placed in enteric transport medium. Collection : Pass the tip of a sterile swab approximately 1 inch beyond the anal sphincter. Carefully rotate the swab to sample the anal crypts and withdraw the swab. Place the swab in transport medium. Transport : • Within 24hrs/4°C. Not recommended for Viruses. No microscopic examination possible. 2/10/2021 Department of Microbiology 48

49. Urinary tract infection(UTI) 1.Urine : Container : Sterile , screw-cap container. Patient preparation: Females : Clean area with soap and water, then rinse with water, hold labia apart and begin voiding in commode; after several ml have passed, collect midstream Males : Clean glans with soap and water, then rinse with water, retract foreskin; after several ml have passed, collect midstream. Collection : After several ml have passed, collect midstream in a urine container. Transport : Within 24hrs/4°C 2/10/2021 Department of Microbiology 49

50. 2.Catheter specimen of urine (CSU ) Container : Sterile , screw-cap container. Patient preparation: Clean urethral area (soap and water) and rinse (water ). Collection : Insert catheter into bladder. Allow first 15ml to pass. Then collect remainder. Transport : Within 24hrs/4°C 2/10/2021 Department of Microbiology 50

51. 3.Suprapubic bladder aspiration It is used primarily for neonates and small children but may be safely used in adults. A full bladder is required for this. Overlying skin id disinfected. Bladder is punctured above the symphysis pubis with a 22-gauge needle on a syringe. About 10ml of urine is aspirated. 2/10/2021 Department of Microbiology 51

52. Abscess Abscess(also lesions, wounds, pustule, ulcer) A . Superficial Abscess: Container : Anaerobic swab moistened with Stuart’s or Amie’s medium Patient preparation: Wipe area with sterile saline or 70% alcohol. Collection : Swab along the leading edge of wound. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 52

53. B . Deep Wound abscess Container : Anaerobic transporter. Patient preparation: Wipe area with sterile saline or 70% alcohol. Collection : Aspirate material from wall or excise tissue. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 53

54. Hair, nails, or skin scrapings (for fungus culture) Container : Clean , screw-top tube. Patient preparation: Nails or skin: wipe with 70% alcohol. Collection : Hair : Collect hair with intact shaft. Nails : Send clippings of affected area. Skin : Scrape skin at leading edge of lesion. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 54

55. Genital tract A . Females: 1.Cervical swab: Container : Swab moistened with Stuart’s or Amie’s medium. Patient preparation: Remove mucus before collection of specimen. Collection : Swab deeply into endocervical canal. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 55

56. 2 . High vaginal swab Container : Swab moistened with Stuart’s or Amie’s medium . Patient preparation: Remove exudates. Collection : Swab secretions and mucous membrane of vagina. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 56

57. 3 . Urethral swab Container : Swab moistened with Stuart’s or Amie’s medium. Patient preparation: Remove exudates from urethral opening. Collection : Collect discharge by massaging urethra against pubic symphysis. OR insert flexible swab 2-4cm into urethra and rotate swab for 2 sec. Collect at least 1 hr after patient has urinated. Transport : Within 24hrs/RT 2/10/2021 Department of Microbiology 57

58. B . Males 1.Prostrate : Container : Swab moistened with Stuart’s or Amie’s medium. OR sterile screw-cap tube. Patient preparation: Clean glans with soap and water. Collection : Collect secretion on swab or In tube Transport : Within 24hrs/RT. 2/10/2021 Department of Microbiology 58

59. 2 . Urethra: Container : Swab moistened with Stuart’s or Amie’s medium. Collection : Insert flexible swab 2-4cm into urethra and rotate for 2 sec. Transport : Within 24hrs/RT for swab 2/10/2021 Department of Microbiology 59

60. Infection control precautions Precautions Use Requirements Contact precautions Patients known or suspected to have serious illnesses easily transmitted by direct patient contact or by contact with items in the patient's environment Gloves Gown Droplet precautions Barrier to stop infections spread by large (>5 microns), moist droplets produced by people when they cough, sneeze or speak Contact precautions Well-fitting mask Eye protection Airborne precautions Patients known or suspected to have serious illnesses transmitted by airborne droplet nuclei Contact precautions Droplet precautions N95 mask Isolation room (In hospital)

61. Expected Questions Describe the specimen collection method for urine specimen collection. Describe the specimen collection method for CSF specimen collection. Describe the specimen collection method for blood specimen collection. 2/10/2021 Department of Microbiology 61

62. Thank You! 2/10/2021 Department of Microbiology 62