Specimen Preparation for LC and LPA Labs

Liquid Culture (LC) and Line Probe Assay (LPA) specimen processing involves pre-treatment of the sputum specimens.

Digestion and decontamination are usually done using N-acetyl-l-cysteine–sodium hydroxide (NALC- NaOH) method: 

• The process is essential to free TB bacilli from the mucus cells/ tissue.
• This also helps in decontamination by killing normal flora that grows more rapidly than the TB bacilli. 

NALC-NaOH Method for Sample Processing
 

Materials required: 
• Disposable 50 ml plastic tubes (Falcon tubes) 
• Sterile NaOH-NALC-sodium citrate solution/ commercial MycoPrep 
• Phosphate buffer pH 6.8 (0.067M)
• Refrigerated centrifuge 
• Vortex mixer 
• Timer 
• Transfer pipettes

Steps:

1. To the sputum sample in the 50 ml centrifuge tube, add equal volume NaOH-NALC-sodium citrate solution.
2. Tighten the cap.
3. Vortex lightly or hand mix (15-30 seconds); keep for 15-20 minutes with mixing/ vortexing gently every 5-10 minutes to completely liquify.
4. Add phosphate buffer (pH 6.8) up to 50 ml mark of tube; mix well (invert mix/ vortex).
5. Centrifuge at 3000 g (15-20 minutes), 4ºC.
6. Wait 5 minutes for aerosols to settle, decant supernatant and discard.
7. Resuspend sediment in 1-2 ml phosphate buffer (pH 6.8).
8. After decontamination,  resuspend the pellet in phosphate buffer (1-1.5 ml) and homogenize samples for proper mixing. This should be followed by the preparation of aliquots.
9. Use aliquot/ sediment to inoculate Mycobacteria Growth Indicator Tube (MGIT) tubes and for DNA extraction for LPA.

Resources

• Guidelines for PMDT in India, 2021. 

Kindly provide your valuable feedback on the page to the link provided HERE