Sputum Liquefaction: from viscous, heterogeneous mucus to equivalent, homogenous samples
Sputum processing has one main purpose; to render the viscous, heterogeneous specimen to a homogenous state (evenly distributed suspension). This homogenous suspension will be dispensed into multiple aliquots for cryopreservation, smear microscopy, gram stain, and culture. Each aliquot should be representative of the original specimen. This can be accomplished through the process of sputum liquefaction.
Sputum is the thick mucus or phlegm that is expectorated from the lower respiratory tract (bronchi and lungs) and is important for the investigation of respiratory disease. It is difficult to process multiple, equivalent samples from Sputum because it is a heterogeneous and viscous substance that varies in appearance (mucoid, purulent, mucopurulent and blood-streaked). Consequently, the various host components and organisms in the sputum are not evenly distributed. Sputum often contains a network of linked mucin molecules that trap microorganisms (such as bacteria) causing them to clump together resulting in unequal distribution. This requires the efficient release of pathogenic bacteria from the sputum’s mucin matrix through the process of chemical and/or mechanical liquefaction.
Chemical liquefaction uses a mucolytic agent to breakdown the mucin matrix to decrease viscosity and release bacteria that may be trapped within the complex sputum network. A variety of mucolytic agents exist, but dithiothreitol (DTT) and N-Acetyl-L-Cysteine (NAC) are most widely used. They are classical mucolytic agents that contain a sulfhydryl group that cleaves the disulphide bonds linking the mucin molecules, which decreases viscosity. Studies have shown that sputum liquefaction with low concentrations of DTT is more effective than NAC with a >90% reduction in sputum elasticity and a greater number or organisms and colony size after culture (1, 3). This may be due to DTT being a dithiol (having 2 redox-active cysteine residues) whereas NAC is a monothiol (2).
Mechanical liquefaction is another method to digest sputum and is often used in conjunction with chemical liquefaction. Sputum can be liquefied by vortexing with the aid of glass beads, magnetic stir bar and stirring plate, or by sonication in the presence or absence of a mucolytic agent. However, mechanical liquefaction is less effective without a mucolytic agent and is usually used to aid chemical liquefaction. For example, cystic fibrosis sputum was not digested effectively with DTT or NAC, but complete homogenization was accomplished when sputum was treated with DTT and sonicated intermittently for 120 seconds (30 second intervals) (4).
To make things more difficult, many samples are contaminated with rapidly growing commensal microbial flora which are normally present in the mouth, throat, etc. It is important to differentiate normal flora and to identify disease-causing (pathogenic) bacteria in a culture. Identification is a step-by-step process that may involve several biochemical, immunological, and/or molecular tests and observations of the organism’s growth characteristics.
Once a homogenous suspension is prepared, it is critical that the sputum is dispensed into cryovials in small amounts (1.5-1.8ml) and is stored at -70°C as soon as possible. Avoid multiple freeze-thaw cycles that could possibly affect either the analyte being measured, or the viability of the pathogenic bacteria.
The sensitivity of sputum testing is largely dependent on the efficiency of the sputum processing protocol to create homogenous, representative samples. G-Biosciences offers Sputo-LR™, which is a concentrated sterile solution of Cleland’s Reagent (DTT) in phosphate buffer at pH 7.0. Sputo-LR™ is provided with a simple and efficient sputum processing protocol, which is effective at liquefying difficult sputum samples. A small amount of DTT is added to the specimen, so it does not affect morphology, growth or fluorescent antibody staining of pathogens in sputum.
- Nielsen H, et al. Elastic Contributions Dominate the Viscoelastic Properties of Sputum from Cystic Fibrosis Patients. Biophysical Chemistry 2004; 112: 193-200.
- Rancourt RC, et al. Thioredoxin Liquefies and Decreases the Viscoelasticity of Cystic Fibrosis Sputum. American Journal of Physiology Lung Cellular and Molecular Physiology 2004; 286: L931-L938.
- Saraswathy VV, et al. Comparative Analysis of Cell Morphology in Sputum Samples Homogenized with Dithiothreitol, N-acetyl-l cysteine, Cytorich® Red Preservative and in Cellblock Preparations to Enhance the Sensitivity of Sputum Cytology for the Diagnosis of Lung Cancer. Diagnostic Cytopathology 2015; 43: 551-558.
- Baxter CG, et al. Homogenisation of Cystic Fibrosis Sputum by Sonication — An Essential Step for Aspergillus PCR. Journal of Microbiological Methods 2011; 85: 75-81.