1. Collecting and transporting sputum specimens
Module 3:
Collecting and transporting
sputum specimens
Global Laboratory Initiative – Xpert MTB/RIF Training Package

2. World Health Organization
Contents of this module
1 April, 2017
Choosing suitable specimen containers
Timing of specimen collection
How to collect a specimen
Handling specimens and referring them for testing
Labelling specimens

3. Learning objectives At the end of this module, you will be able to:
Describe specifications of suitable containers for sputum collection
Discuss different sputum collection strategies: spot/morning/spot, spot/spot, single spot for Xpert MTB/RIF
Describe and demonstrate safe and correct collection of sputum
Describe options for specimen collection, handling and transport
List features of a good quality sputum specimen
Outline requirements for a properly labelled specimen

4. Sputum-specimen collection: container specifications
Adapt according to the NTP’s guidelines in your country
30-50 ml capacity
Translucent or clear material
Sides and walls that allow easy labelling
Single-use combustible material
Leak-proof with a screw-cap
Wide mouth

5. Sputum specimen collection: timing
World Health Organization
1 April, 2017
Sputum specimen collection: timing
Spot–morning–spot
WHO and The Union recommend this timing:
Spot In the clinic – during the initial visit
Early morning At home – first sputum produced in the morning on the day of the second visit to the clinic (ideally, the day after the initial visit)
Spot In the clinic – additional sputum collected during the second visit.

6. Collection strategies: spot-morning-spot
Advantages
A spot specimen is available in case the patient does not return with the morning specimen.
Disadvantages
Requires two visits to the clinic.
Diagnosis takes at least 2- 3 days.
Increases the workload.
There is a high risk of missing a case if only the first specimen is received.

7. Collection strategies: WHO’s revised policy
WHO’s policy on case detection by microscopy was revised in 2007:
The recommended number of specimens examined was reduced from three to two in countries with appropriate external quality assurance (EQA) procedures and documented good quality microscopy
In these settings, the case definition was also revised to one AFB positive smear, defined as one or more acid-fast bacilli in at least 100 microscopic fields
If appropriate quality assurance is not available, three specimens should be examined.

8. Same-day strategy: spot-spot
Good quality microscopy of two consecutive sputum specimens (spot-spot) identifies 95–98% of smear- positive TB patients.
Advantages – this strategy:
Greatly reduces the workload of laboratories
Has the potential for offering same-day diagnosis
Is better for patients because it reduces the number of visits while largely maintaining sensitivity.
Disadvantages:
Very minor loss in the number of cases detected.

9. Sputum collection: overview
Follow the NTP’s guidelines on sputum collection.
Ensure that sputum collection is supervised.
A single sputum specimen is recommended for Xpert MTB/RIF:
Increased yield is obtained by testing multiple specimens but this increases the cost of testing
An additional sputum specimen may be needed in case of an error or invalid Xpert MTB/RIF result
At least 1 ml of sputum should be collected.
Additional sputum specimens may be needed for microscopy, culture and DST, depending on the NTP’s guidelines (Xpert MTB/RIF is not recommended for monitoring patients’ treatment).
Avoid using pooled sputum specimens.

10. The NTP’s guidelines for sputum collection in (country name)
[Insert the NTP's specific guidelines]
To be customized by each country

11. Sputum collection: safety
When providing a sputum specimen, a patient may produce infectious aerosols and therefore biosafety precautions are needed:
Instruct the patient to cover his or her mouth when coughing
Never collect sputum in the laboratory
Collect sputum away from other people in a well ventilated space following the NTP’s guidelines
Do not stand in front of the patient during specimen collection!

12. Sputum collection: Patient education and instructions
The best specimen comes from the lungs.
Saliva or nasal secretions are unsatisfactory.
Specimens should not contain food or other particles because the test may not work properly.
Patients should be instructed to take the following steps to produce the best specimen:
Wash your mouth with clean water to remove food and other particles
Inhale deeply 2–3 times and breathe out strongly each time
Cough deeply from your chest to produce sputum
Place the open container close to your mouth to collect the specimen; do not get sputum on the outside of the container
Wash your hands after collecting the sample.

13. Specimen quality: Optimal quality
Mucoid
Purulent
(Images courtesy of A. Van Deun)

14. Specimen quality: Suboptimal quality
Bloodstained
Salivary
(Images courtesy of A. Van Deun)

15. Specimen quality: overview
Obtaining an adequate quantity of good quality sputum is critical to ensuring accurate test results. For best results, obtain 1-4 ml of purulent/mucoid sputum.

16. Request for sputum examination
A request for sputum examination should include the:
Name of the treatment unit
Date of the request
Patient's information (that is, name, sex, age, address and the register number of the patient or suspected case)
Number of specimens and types of specimens sent for testing
Date the specimens were collected
Reason for examination (for example, diagnosis or follow-up)
Signature of the person requesting the examination.

17. Laboratory request form (example) (1 of 2)
To be customized by each country.
Add your local request form, and show participants how to correctly complete the form.

18. Laboratory results form (example) (2 of 2)
To be customized by each country.
Add your local request form, and show participants how to correctly complete the form.

19. Labelling the specimen container
Label with the patient’s name, identification number and the date of collection.
Label the outer sides of the container with permanent ink.
Never label the lid.

20. Receiving specimens in the laboratory
Check the quantity and quality of specimens:
Check the volume (ideally 1-4 ml; a minimum of 1 ml is required for Xpert MTB/RIF testing)
Record sputum consistency (mucoid, purulent, bloody or watery) on the report form
Ensure that specimens do not contain food or other particulates.
Check that the patient’s information is complete, and that Xpert MTB/RIF testing has been requested in accordance with the NTP’s guidelines.
Follow the NTP’s guidelines for rejecting samples.
Ensure that the patient’s information is complete on both the request form and the specimen container, and ensure that the information matches.
Record the specimen in the laboratory register and allocate a laboratory serial number to the specimen.

21. If patients are referred If specimens are referred
Referral systems
Peripheral facilities that do not perform Xpert MTB/RIF testing should refer either patients or specimens to a testing laboratory.
If patients are referred
If specimens are referred
Advantages
Sputum collection can be monitored and collection can be repeated if the first specimen is not satisfactory.
Treatment can be initiated faster.
Patient does not have to travel.
Disadvantages
Requires patients to travel.
Requires systems for safe transport and shipment.
Requires efficient system for transmitting results.
Specimen transport may require cold-chain or preservatives.

22. Specimen referral
The shipment of specimens constitutes a public-health risk, and the shipper is responsible for ensuring that samples are packed safely.
Triple packaging (described in later slides) must be used:
Locally appropriate solutions can be used as long as safety requirements are met.
Country-specific safety guidelines must be followed in order to correctly categorize the specimen and its associated risk (eg, Category A or B, as per WHO Guidance on regulations for the transport of infectious substances, 2013–2014).
To be customized by each country

23. Transporting specimens
To be customized by each country
Specimens should be transported to a laboratory as quickly as possible:
A sputum specimen that has been delayed during transport can still be used for microscopy and Xpert MTB/RIF testing.
Whenever possible, specimens should be refrigerated at 2–8°C for a maximum of 10 days. However, if necessary, specimens can be stored at a maximum of 35°C for up to 3 days, and then refrigerated at 2–8°C for a combined maximum duration of 10 days.
The total number of specimens in a box must correspond to the number of accompanying request forms.
The identification number on each sputum container must correspond to the number on a request form.
The accompanying forms must contain the required information for each patient.

24. Triple packaging: Step 1 of 3
Primary packaging
Wrap the leak-proof container in cotton wool or paper towels in a sufficient quantity to absorb the entire contents in case of leaks.

25. Triple packaging: Step 2 of 3
Secondary packaging
Place the wrapped container inside a secondary container, such as a self- sealing plastic bag or another container.
Place the secondary container in a rack to prevent leakage.

26. Triple packaging: Step 3 of 3
Tertiary packaging
Place the secondary container and its contents in an approved safety cooler box or another appropriate container in an upright position.
Place a biohazard sign – with markings and labelling appropriate for the specimen category – on the tertiary container.

27. Role play: Sputum collection
Purposes
To practise educating a person suspected of having TB about the importance of a properly collected sputum specimen and to practise providing instructions on how to collect a specimen
To practice packaging sputum specimens for referral.
Total time
40 minutes.
Process
Work in groups of 4.
Choose roles: health worker, person suspected of having TB, dispatcher and observer.
The health worker instructs the person suspected of having TB on how to produce a specimen, and then provides the specimen to the dispatcher for packaging; the observer provides feedback.
Switch roles and repeat the process until everyone has played each part once.

28. Role play: Debrief
Did the health-care worker follow appropriate procedures?
COMPLETE a Request for Sputum Examination form
LABEL the sputum containers
EXPLAIN to the person suspected of having TB how to collect sputum and the importance of a properly collected specimen
ASK the person suspected of having TB to produce a first sample
ASK the person suspected of having TB to collect another sample the next morning and then bring it to the diagnostic centre (OR modify the advice to follow the NTP’s guidelines)
PACKAGE appropriately the sputum sample for referral.
Did the person suspected of having TB understand the instructions?
Did the dispatcher receive the package appropriately and complete the administrative procedures correctly?
What suggestions for improvements did the observer note?

29. Summary
Not all containers are suitable for specimen collection: follow the NTP’s guidelines on the size or volume of the container to be used, and the material and type of lid.
An adequate quantity (1-4 ml) of good quality sputum (that is, purulent/mucoid) as well as a properly completed Laboratory Request Form and properly labelled specimen containers are critical to ensuring accurate results from the Xpert MTB/RIF test.
Patients should be educated on how to produce good quality specimens, and sputum collection should be supervised.
A well functioning referral system (for patients or specimens, or both) is critical to ensuring a high quality diagnostic service: follow country-specific safety guidelines for storing and shipping samples; triple packaging must be always used.

30. Assessment
What are the the NTP’s guidelines for collecting sputum specimens in your country?
Why should sputum never be collected in a laboratory?
What are the options for referral at peripheral health centres that do not perform the Xpert MTB/RIF test?
What are the features of a good quality specimen?
How should a sputum container be labelled?
What supplies are needed for triple packaging for TB diagnostic samples?