TrueNAT Sample Processing: Other Body Fluids

The extra-pulmonary samples (Non-sputum) can also be processed and used for MTB and Rif diagnosis using Truenat. The processing of various extra-pulmonary samples is described below.

Reagents required:

Trueprep AUTO MTB Sample Pre-treatment Pack for sample processing

• Reagents stable for two years (2-40°C) or one month (temperatures up to 45°C)
• Avoid exposure to light or elevated temperatures
• Do not freeze

Steps in processing of non-sputum samples

Non-sputum samples like tissue biopsy should not be processed at the Designated Microscopy Centre (DMC), but should be transferred to a higher centre.

General Instructions

1. Disinfect work surfaces with freshly prepared 1% bleach, followed by 70% alcohol.
2. Process fresh specimens immediately or store frozen at -20^oC. Avoid more than three freeze thaws.
3. Bring frozen samples and refrigerated reagents to room temperature (20-30°C).
4. Open the Trueprep AUTO MTB sample pre-treatment pack that contains:
   • Liquefaction buffer bottle
   • Lysis buffer bottle
   • Graduated 1 ml transfer pipette.
5. Processing is to be done as instructed for each sample type.
6. Non-sputum sample is stable for 3 days at up to 40°C and 1 week at 30°C in the lysis buffer.

A. Processing for Bronchoalveolar Lavage (BAL), Pleural fluid, Peritoneal fluid 

Equipment: Centrifuge and centrifuge tubes (10-15 ml volume)

1. Add 5-10 ml specimen in a centrifuge tube.
2. Centrifuge at 4000x for 5 minutes to concentrate the sample.
3. Discard the supernatant until 500 μl remains.
4. Add two drops of the liquefaction buffer to the concentrated sample (500 μl).
5. Transfer the contents to a labelled lysis buffer tube.
6. Incubate for five minutes and proceed for DNA extraction.

B. Processing for Pus, Abscess, Lymph node aspirate, Cerebrospinal Fluid (CSF)

Equipment: Centrifuge tubes (1.5 ml volume)

1. Add 0.5 ml specimen in a 1.5 ml tube.
2. Add two drops of liquefaction buffer.
3. Transfer the contents to a labelled lysis buffer tube.
4. Incubate for 5 minutes and proceed for DNA extraction.

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C. Processing for Tissue/ Biopsy Samples 

Equipment: Mortar and pestle (for tissue homogenization)

1. Homogenize sample by using 100 μl lysis buffer in mortar and pestle.
2. Add two drops of liquefaction buffer.
3. Transfer the contents to a labelled lysis buffer tube.
4. Incubate for five minutes.
5. Use only clear fluid for DNA extraction.

The processed samples will be used for DNA extraction and PCR amplification for diagnosis of MTB and Rif resistance.

Resources

• Trueprep AUTO MTB Sample Pre-treatment Pack.

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