# Evaluation of a Particle Enhanced Turbidimetric Immunoassay (PETIA) for detecting calprotectin in sputum samples

## TL;DR

This study tests a new method to measure calprotectin in sputum samples, finding it reliable and stable for up to two months when stored properly.

## Contribution

The study introduces a validated particle enhanced turbidimetric immunoassay (PETIA) for calprotectin in sputum with proven stability and precision.

## Key findings

- Calprotectin measurements in sputum using PETIA showed high precision with a coefficient of variation below 3%.
- Calprotectin levels remained stable in sputum samples stored at 4°C for up to one week and at -20°C for up to two months.
- Spiking and dilution studies confirmed the accuracy of the PETIA method with deviations below 10%.

## Abstract

Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are characterized by persistent airway inflammation and airflow limitation. Measuring airway inflammation may be of value both in research and clinical practice. Calprotectin, a protein associated with neutrophil activation, serves as a potential biomarker for airway inflammation. This study evaluates the performance of a particle enhanced turbidimetric immunoassay (PETIA) for measuring calprotectin in sputum samples.

Thirty sputum samples from unique patients were collected at the Department of Clinical Microbiology in Lund, Sweden and treated with dithiothreitol (DTT) before analysis. Calprotectin levels were measured using a PETIA on an automated turbidimetric analysis system (Atellica CH 930 Analyzer). The 30 samples were analyzed in triplicate the day of sample collection (Day 0), after storage at 4 °C for two to four days (Day 2–4), and after six to seven days (Day 6–7). An additional aliquot from each sample was stored at -20 °C and analyzed after 41 to 58 days (Day 41–58). Precision, accuracy, and stability of the assay were assessed. Precision was evaluated using pooled- and individual sputum samples while accuracy was determined through spiking studies and dilution linearity. Stability was tested by comparing calprotectin levels Day 2–4, Day 6–7 and Day 41–58 with Day 0, respectively. Instability equations were calculated for samples stored at 4 °C and at -20 °C, respectively.

Three different pools were each analyzed 25 times during five days. The coefficient of variation (CV) for the calprotectin concentration was below 3% for all three. Considering precision based on individual samples, 90% of 120 triplicates had a CV below 7%. These figures indicate an acceptable precision. The spiking and dilution studies showed that measured calprotectin concentrations matched expected levels, with deviations below 10%. Calprotectin concentrations Day 2–4, Day 6–7 and Day 41–58 did not significantly differ from Day 0, and the slope of the instability equations did not significantly differ from 0. It implies that calprotectin concentration can be measured in DTT treated sputum stored for up to one week at 4 °C and up to two months at -20 °C.

The evaluated PETIA, running on an automated system, provides a promising method for measuring calprotectin in sputum, supporting its potential use in both clinical and research applications.