STLS: Collection & Storage of Good Quality Specimen

Three things are received by the Lab Technician- Sample for testing, request form, and  request for test in Nikshay.

Accepting the request for testing includes the following steps:

1. The LT verifies details on the request form that has eleven parts.

• The first part contains details on the name of referring facility, name of the patient, complete address, age & gender of the patient, date of referral, type of presumptive TB, the key population to which the patient belongs and site of disease.
• The second part contains details of referring facilities, Nikshay ID, and the names of State, district and TB units.
• The third portion is for the diagnosis and follow-up of TB.
• The fourth portion is for the diagnosis and follow-up of drug-resistant TB.
• The fifth portion is to indicate the required tests with the details of the person requesting the test.
• Parts six to eleven are used for reporting test results.

2. The LT verifies the quality of the sample received.

3. LT captures details on the patient, reasons for testing, test requested, and the visual appearance of the sample in the TB Laboratory register.

4. LT verifies the test request generated in Nikshay against the test ID requested (Figure).

Figure: Test Details Added in Nikshay by the Referring Health Facility; Source: Guidelines for PMDT in India, 2021.

5. LT initiates the test requested.

Resources

• Guidelines for PMDT in India, NTEP, 2021.
• Training Modules (1-4) for Programme Managers and Medical Officers, NTEP, 2020.

Assessment

Presumptive pulmonary TB patients are subjected to sputum smear microscopy (Ziehl Neelsen (ZN)/ Florescence Microscopy (FM)). Two consecutive sputum specimens will identify the vast majority (95–98%) of smear-positive TB patients

Two specimens are collected:

• One Spot and one early morning sample OR
• Two supervised spot specimens collected at least one hour apart, and smears made from both the samples.

If one or both smears are positive, the patient is diagnosed as a microbiologically confirmed pulmonary TB case.

• The spot specimen collected is labelled as 'a'.
• While the patient is given a labelled container with instructions to cough out sputum into the container early in the morning after rinsing the mouth with water. This is the early morning specimen. This is labelled as specimen 'b'.

• If the health facility is not a Designated Microscopy Centre (DMC), then the patient is given a sputum container with the instructions to collect an early morning specimen and go with the sputum specimen to the DMC where the spot specimen can be collected.
• In case the patient is not able to travel to the DMC, then the spot specimen could be collected at the nearest health facility or sputum collection centre and transported to the DMC.
• These two samples should be collected within a day or two consecutive days.
• Two supervised spot samples may be collected one hour apart if patient is too sick, coming from a long distance or likelihood of not giving a second sample is significant.
• Collection of early morning specimens is preferred because of the overnight accumulation of secretions. However, spot samples collected at any time for patients is also suitable if productive sputum is expectorated after deep cough.

Resources

1. Module for Laboratory Technicians.
2. Training Modules for Programme Managers and Medical Officers.

Assessment

Educating patients on collection is essential to have good quality sputum. The healthcare worker (HCW)/ medical officer (MO) or the laboratory technician (LT) can educate patients on how to collect and dispense sputum.

The HCW/MO/LT provides a new sputum cup with the Laboratory Serial Number written on its side to the patient. They should explain that sputum should be collected in an open place or in a well-ventilated room; it should not be collected in closed rooms, toilets and ill-ventilated rooms

A specimen collected under supervision is likely to yield better results. Supervising person has to demonstrate how to collect good sputum and dispensing it:

1. Using a laboratory sputum cup, demonstrate how to open the lid of the specimen container and place it conveniently within their reach, so they can close it immediately after collecting sputum and also how to screw the cap on the cup tightly so it doesn't leak.
2. Demonstrate how to bring up sputum, beginning with rinsing their mouth as food particles may give false positive results.
3. Demonstrate deep inhalation (2–3 times) and let the patient know that this will initiate the cough reflex in most individuals.
4. Demonstrate how individuals can place their palms on the waist, squat or sit and continue deep breathing again. Tapping or thumping of the back 
   may encourage expectoration (Sitting and placing hands on the waist fixes the shoulder and pelvic muscles and brings the intercostal muscles of ribcage and diaphragm into action)
5. After deep inhalation and coughing deeply, the sputum should come up in their mouth. The sputum is retained in the mouth and allowed to fall from their tongue into the pre-labeled container. Patient should be encouraged not to spit into the container. The patient can also be encouraged to cough directly into the cup.
6. The patient’s mouth should not touch the container and the patient must ensure that sputum does not touch the outside of the container.
7. The patient should not open the sputum cup till they are ready to use it
8. They should not rub off the number written on the side of the container
9. They should not touch the inside of the container with their fingers or tongue while collecting sputum

The person collecting the specimen should make sure that no one stands in front of the individual who is trying to cough up the sputum. When an individual has only coughed up saliva or has not coughed up at least 2 ml of sputum, they should be encouraged to give good specimen that is of appropriate quantity.

Resources

• Module for Laboratory Technicians
• Training Modules for Programme Managers and Medical Officers

Assessment

The Healthcare Worker (HCW) needs to carefully explain how to collect a good quality sputum specimen. He/she needs to demonstrate how to bring up sputum from the chest, what a good sputum specimen looks like, and the quantity of sputum required.  

Characteristics of a Good Sputum Sample 

• Thick (semi-solid) muco-purulent (yellowish) in consistency, coughed out deeply from the lungs
• Sufficient in amount i.e., 2 to 5 ml (or enough to cover the size of a fingernail at the bottom of the container)
• It should not be blood-stained (brownish colour) as far as possible.

Steps to Ensure Good Quality Sputum Sample

1. Explain to the patient the characteristics of sputum - that it is thick and mucoid as compared to saliva which is thin and watery.
2. The patient should preferably rinse his/her mouth to get rid of any food particles which may give false-positive results.
3. One should demonstrate to the patient by action how s/he should take deep breaths and bring up the sputum.
   1. The patient is instructed to inhale deeply (2–3 times), which will initiate the cough reflex in most patients.
   2. The sputum is retained in the mouth and spit into the pre-labelled container without spilling.
4. Some patients may not be able to expectorate with deep breathing, in which case HCW should demonstrate to them how they should place their palms on the waist, squat or sit and continue deep breathing again.
   1. Tapping or thumping of the back may encourage expectoration. (Sitting and placing hands on the waist fixes the shoulder and pelvic muscles and brings the intercostal muscles of the ribcage and diaphragm into action).
5. When a patient has only coughed up saliva or has coughed up less than 2 ml (the size of a fingernail at the bottom of the container) of sputum, the patient should be encouraged to provide a better specimen.

Resources

• RNTCP Module for Laboratory Technician, CTD, MoHFW, 2005.

Biological specimen/ samples collected on reaching a TB testing laboratory needs to be formally received. The sample may be handed over by agents(couriers, health staff/ volunteers, patient representatives) or by patients themselves. The formal receipt of sample enables further processes such as testing and communication of results back to the patient. If the sample is successfully received, the appropriate testing process is initiated using the sample, else it is rejected and a fresh sample requested. 

The designated Lab Technician (LT) at the Laboratory is responsible for receiving the sample. To initiate the receipt, the sample along with the patient, or the sample along with accompanying test request is required. If it is available, following are the steps to complete sample receipt:

1. Compare patient information (Patient Name and Patient ID / Sample ID/ Test ID) to the test request that has been made.
   • This is performed by searching the patient ID in Nikshay under the Diagnostics menu and comparing and matching the patient details and the label on the sample.
   • If a physical form (Request for examination of Biological specimen for TB) is available, the details on the sample label and form needs to be compared and matched as well.
2. If the details are matched the LT then checks the quality (mucopurulent, non-blood stained), quantity (adequate to perform the requested test) of the sample and ensures that there is no leakage.
3. If the above checks are passed, then the LT Accepts sample. If not the LT rejects sample with a request to get a new sample. The rejection of the sample and request to obtain a new sample is recorded on Nikshay and is communicated to the patient and the person who requested the test.  
4. To register the receipt by accepting/Check in the sample for testing in Nikshay. The relevant information may also be captured in the TB Laboratory Register.  Ensure that the Test ID (if not already present) / Lab serial number from the Lab register is labelled on the sample container.

Sample receiving in Nikshay

Figure: Sample Journey Tracking in Nikshay; Source: Nikshay Diagnostic Training Content.

Resources

1. Training Modules (1-4) for Programme Managers and Medical Officers, NTEP, 2020.
2. Nikshay Zendesk, Nikshay Knowledge Base, FAQs.
3. Nikshay Zendesk, Nikshay Knowledge Base, Diagnostics.

Assessment

Storage conditions of sputum sample can effect the test results.

• Sputum samples should be transported to the laboratory as soon as possible after collection.
• It is the responsibility of Laboratory Technician (LT) and Senior TB Laboratory Supervisor (STLS) to ensure proper storage and transport of sputum specimens.
• Sputum is stored to preserve the specimen quality.
• The stored sputum samples should not be frozen.

Storage of Sputum Samples

For microscopy

• For smear microscopy, sputum specimens should be examined on the same day and not later than 2 days after collection.
• If delay is unavoidable, the sputum collected should be stored in a cool place/ refrigerated at 4°C to inhibit the growth of unwanted microorganisms.
• Stored sputum samples should be protected from light and heat to prevent liquefaction of the sample, else it makes the selection of mucopurulent part of the sample difficult.
• Samples received over holidays/weekends should be stored in a cool place/ refrigerated at 4°C.

For liquid culture

• Sputum should be stored in a cool place/ refrigerated at 4°C to inhibit the growth of unwanted microorganisms; not later than 3 days after collection.

For molecular tests - Nucleic Acid Amplification Test (NAAT) and Line Probe Assay (LPA)

• Sputum must be stored by refrigerating at 4°C to inhibit the growth of unwanted microorganisms and transported in cool chain to the nearest molecular laboratory. It should not be stored beyond one week at 4°C.

Resources

Technical and Operational Guidelines; Chapter 3: Case finding and diagnosis strategy

PMDT Guidelines 2021

Assessment