2. Module 5 Collection, transport and receipt of specimens 1

3. Learning objectives At the end of this module you will be able to:  explain the proper collection of specimens;  explain how to select suitable containers for the collection of different specimens;  explain the proper labelling of specimens to be submitted to the TB laboratory;  explain the proper transportation of specimens;  list the features of a good specimen;  fill in the laboratory register. 2

4. Content outline Procedure for specimen collection Suitable specimen containers Transport of specimens Receipt of incoming specimens Evaluation of specimen quality Specimens and request forms: quality assurance 3

5. REMEMBER! Unless specimens are collected with the utmost care and promptly transported to the laboratory under the proper conditions, the advantages of culture will not be fully realized. If sputa are stored at 4°C, delay between collection and inoculation should not exceed 7 days. 4

6. Collection procedures for sputum specimens SPOT MORNING SPOT Minimum volume: 2 ml (optimal volume: 5 ml) New WHO policy: two samples (better if one is a morning sample). Can be adopted where good quality assurance programmes are in place (good performance documented) in countries with high workloads and limited human resources. 5

7. Collection procedures for other specimens Specimens from sterile sites, aseptically collected. Specimens from non-sterile sites, specimens expected to be contaminated by normal flora, or specimens not collected under sterile conditions. 6

8. Other respiratory specimens Bronchial aspirate (2–5 ml). Bronchial alveolar lavage (20–40 ml). Transbronchial biopsies, brushing (add 0.5–1 ml sterile saline to avoid drying). Pleural effusion (20–50 ml). Pleural biopsy Gastric aspirate (early morning, empty stomach, transport immediately to the laboratory, neutralize by adding 1–2 ml of 10% trisodium phosphate solution, depending on the specimen volume or 100mg of sodium bicarbonate): for children, or people unable to produce sputum. 7

9. Contaminated specimens: instructions for urine collection External genitalia should be washed before specimen collection. A single early-morning urine specimen (approx. 200 ml) should be collected. Specimens should be refrigerated or transported immediatelyto the laboratory. 8

10. Aseptically collected fluids Body fluids: – spinal – pleural – pericardial – synovial – ascitic – blood (5–10 ml citrate blood) – sperm and prostate secretion (no addition) – pus – bone marrow. 9

11. Aseptically collected fluids: additives? Sterile containers without fixatives or preservatives must be used. If clots are expected, add an anticoagulant, e.g. potassium oxalate (0.01–0.02 ml of 10% neutral oxalate per ml fluid), or heparin (0.2 mg/ml), or sodium citrate (two drops of 20% sodium citrate for every 10 ml of fluid). 10

12. Aseptically collected tissues Keep at 4–15 °C. Avoid drying. Deliver specimens to the laboratory as quickly as possible. 11

13. Proper containers for sputa Robust Leakproof Clean 50-ml capacity Translucent or clear material Single-use combustible material Wide-mouthed, screw- capped with a watertight seal Easily-labelled walls 12

14. Preservation methods for sputum samples (1) Sputum samples should be delivered to the laboratory without any delay. When samples cannot be processed on the day of collection, they must be stored in the refrigerator and may be kept for up to 7days. If refrigeration is not available, use of preservatives should be considered. 13

15. Preservation methods for sputum samples (2) Two methods provide reasonable results: 1.Mix the fresh specimen with an equal volume of 1% cetyl pyridinium chloride (CPC) in 2% sodium chloride, but: –centrifuge specimen twice before culturing; –use only egg-based media; –do not inoculate liquid media; –the smear may be more difficult to be made and read. 2.When culture will begin within less than 24 hours, specimens may be mixed with an equal volume of 10% trisodium phosphate. 14

16. Transport of infectious substances Basic triple packaging system (i) a leak-proof primary receptacle(s); (ii) a leak-proof secondary packaging containing sufficient additional absorbent material shall be used to absorb all fluid in case of breakage For cold transportation conditions, ice or dry ice shall be placed outside the secondary receptacle. Wet ice shall be placed in a leakproof container. (iii) an outer packaging of adequate strength for its capacity, mass and intended use. 15

17. Transport of infectious substances 16 Category A Category B

18. Transport of TB infectious substances Classification of infectious substances for the purposes of transport, as per TB: Category A: Cultures of M. tuberculosis. For surface transport, when cultures are intended for diagnostic or clinical purposes, they may be classified as category B. For surface transport there is no maximum quantity per package. Category B: Other infectious substances. Guidance on regulations for the transport of infectious substances 2007–2008. Geneva, World Health Organization, 2007 (WHO/CDS/EPR/2007.2) available at http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2007_2/en/index.html). 17

19. Surface transport : Example of a secondary packaging 18

20. Air transport For national flights (within one country) national civil aviation authorities apply national legislation. For international flights: International Air Transport Association (IATA) www.iata.org 19

21. Transport: national regulations [To be customized according to the country’s specific rules for transport of infectious substances affecting humans.] 20

22. Receipt of incoming specimens 1.Inspect the delivery box for signs of leakage. 2.Disinfect the outside of the delivery box. 3.Open carefully and check for cracked or broken specimen containers. 4.Disinfect the inside of the box. 21

23. Identification of specimens before processing – critical issues Request forms must be separate from specimen containers. Each container should be clearly labelled on the side – not on the cap. Accompanying list should include the requested data for each patient. The number of specimen containers in the box must correspond to the number of names on the accompanying list. The identification number on each specimen container must correspond to the identification number on the accompanying list. The date of dispatch and the name of the health centre must be included on the accompanying list. 22

24. Laboratory request forms for culture and DST 23

25. Specimens and request form: quality assurance Perform tests only upon written request. Insist on specimen request forms being kept separate from the specimens themselves. Insist on properly completed request forms and proper labelling of specimens to ensure positive identification of patients. Reject specimens that cannot be properly identified. Document the arrival time of specimens in the laboratory and note any delays in delivery on the report form; this is particularly important in the case of negative/contaminated results. 24

26. Laboratory register : culture../..

27. Specimen quality: sputum* Morphological characteristics of a good sputum specimen: –minimum amounts of nasal discharge; –saliva is not acceptable; –mucoid or mucopurulent aspect; –volume of 3–5 ml. * Report visual aspect and volume. Avoid pooled sputa – but if you receive them, culture them! 26

28. Specimen quality: other respiratory specimens Volume, delay in processing, and contamination are the critical parameters. Bronchial secretion (2–5 ml). Bronchial aspirate lavage (20–40 ml). Pleural effusions (20–50 ml). Transbronchial and other biopsies : avoid dryness (small biopsies may dry out easily). 27

29. Specimen quality Reject specimens in the following cases: –inadequate volume submitted;* –dried swabs; –pooled urine; –broken containers; –specimens stored and/or transported in improper conditions. *or, process and report volume as insufficient. 28

30. True and false exercise 1.Most samples examined in a TB laboratory are sputum samples. 2.Different samples may require different processing procedures and transport time/conditions. 3.The amount of sample does not affect the sensitivity of TB cultures. 4.Sputum samples can be kept refrigerated for 48 hours. 5.Samples containing CPC need to be maintained at room temperature. 29

31. Module review: take home messages  Good quality specimens are the cornerstone for high quality tuberculosis diagnosis.  The quality of incoming specimens should be evaluated and recorded.  Proper collection procedures and containers, adequate volumes and appropriate specimen transport conditions can all affect TB culture results.  Packaging of infected specimens that are to be sent by air or surface mail must be carried out according to national biosafety guidelines or international rules.  Correct labelling of specimens is critical for their identification. New WHO policy: The number of specimens for TB case diagnosis has been reduced from 3 to 2 in countries with high workloads where microscopy is performed under successful quality assurance programmes. 30

32. Self-assessment Describe the spot–morning–spot strategy. What are the specifications forsuitable containers for specimens collected from different body sites? Describe the requirements for a properly labelled specimen. What are the features of good- and poor-quality specimens? Why and when is it necessary to add preservatives to specimens? 31