WHY sputum
Direct airway biology. Difficult chemistry.
Sputum gives direct access to the airway’s cells and inflammatory proteins. But from the moment it’s collected, the sample works against the assay.
Mucin
Makes samples too thick to pipette accurately.
Proteases
Break down the proteins you want to measure.
Cell debris and saliva
Vary widely between people, and between samples from the same person.
Saline induction
Adds an unknown dilution, so samples from different collection methods can’t be directly compared.
THE CHALLENGE
Why the usual fixes fall short
DTT can damage the assay
Dithiothreitol (DTT) is routinely added to make sputum workable. But it breaks disulfide bonds, and antibodies depend on them. The step that makes sputum easier to handle can undermine the assay that measures it.
Dilution loses the signal
Diluting the sample also makes it easier to handle, but pushes low-abundance markers below the limit of detection.
There is a better way to approach sputum.
our approach
Analysed neat
Measuring sputum as it is
Published feasibility data shows sputum can be profiled neat: undiluted, with no viscosity correction, so no signal is lost to make the sample workable. More than 150 of 250 inflammatory proteins were detected.
1. Barcoded antibodies
Each antibody pair carries a DNA tag.
2. Capture and wash
Mucus, debris and interfering material are washed away before measurement, so the antibodies don’t have to survive the raw sample.
3. Ultra-sensitive readout
Counting the barcodes detects proteins at attomolar levels from microlitre volumes.
What that means in sputum specifically
Alamar's published detectability data puts sputum at more than 150 detectable targets on the Inflammation Panel 250, over 60% of the panel above the limit of detection.
It also means roughly 40% of the panel will not clear the limit of detection in sputum, and which 40% depends on your disease biology and your collection method. Working out whether your specific markers sit in the detectable group is the first thing we do.
A dirty, precious, low-volume sample is the case this chemistry was designed for.
We will confirm performance for your markers, in sputum collected under your protocol, with your DTT position, through a pilot before you commit a cohort. If DTT compromises your targets, you will find out on one plate.
what to use
What you would measure
Sputum work centres on immune profiling of the airway: cytokines, chemokines and mediators of neutrophilic and eosinophilic inflammation, from a single sample. Which panel fits depends on the question.
The right platform for the question
We are one of only five laboratories worldwide certified for NULISA custom assay development, and the UK's first Alamar Certified Service Provider for NULISA™.
We run the same assay, instrument and chemistry here in the UK, under HTA licence.
NULISA
For broad, ultra-sensitive profiling from microlitre inputs, reported in NPQ units as relative quantification. Published sputum detectability is over 60% of the Inflammation Panel 250.
Simoa® or MSD®
Where a study needs absolute quantification in pg/mL for a defined marker set.
We will tell you when that is the better answer.
Sample requirements
Sample type: processed sputum supernatant
Volume to ship: set by the assay rather than by the matrix. Ship what your assay requires plus roughly 10 µL of dead volume, so a repeat stays possible. Sputum is a specialist matrix, so we confirm the usable volume of supernatant after processing at scoping
Processing: plug selection or whole expectorate, whichever your protocol uses. We have no preference between the two. Homogenise, centrifuge, retain supernatant, and keep the choice consistent across the study
DTT: tell us whether DTT was used, at what concentration and at what stage. We do not require it either way, but we do need to know, and it should be consistent across the study
Protease inhibition: consider it at processing, given sputum protease activity
Aliquoting: single-use aliquots, to avoid repeat freeze and thaw
Storage: −80°C
Shipping: on dry ice, with a manifest listing sample ID, volume, induced or spontaneous, collection date, DTT status and freeze and thaw history
These are recommendations. If your collection protocol is already validated, tell us what it is and we will design the work around it.
We confirm receipt and condition in writing on the day samples arrive, before anything is consumed.
Shipping samples internationally
We can arrange collection and shipping, or work with your preferred courier. Either way we confirm the logistics, temperature control and documentation at scoping, before any samples move.
A workflow defined for sputum
Quality and sample integrity
Documented SOPs
Full sample chain of custody
GCP-compliant workflows
HTA licence 12818, the UK framework governing the storage and use of human tissue
Aligned to ISO 15189, the international standard for medical laboratories
Sputum degrades quickly and unevenly, so chain of custody is not a formality here. We record receipt condition, temperature on arrival and freeze and thaw history, and we tell you when a sample arrives in a state that will limit what the data can support.
For research use only. Not for use in diagnostic procedures.
Discuss your matrix with a scientist
Send us your matrix, volumes, normalisation approach and the markers that matter.
We will tell you what is feasible, which platform fits and how long it will take, before you commit anything. If it cannot be done, that is what you will hear.