Optimization of the conditions for preservation of induced sputum: influence of freezing on cellular analysis.

BACKGROUND

Induced sputum (IS) analysis is a noninvasive, valid, and reproducible method for evaluating airway inflammation. It has been suggested that freezing of IS samples in order to delay analysis is feasible. However, the optimal conditions for preservation of IS samples have not been determined.

OBJECTIVES

To determine optimal freezing conditions of IS samples, ensuring adequate specimen quality for assessment of cell viability, total cell count, and differential cell count.

SUBJECTS AND METHODS

Twenty-one subjects were enrolled: 6 healthy control subjects, 5 patients with allergic rhinitis, 5 patients with mild asthma, and 5 patients with severe asthma. Each came to the laboratory once for IS sampling. Cell plugs were homogenized with dithiothreitol and separated into 12 aliquots. Viability and total and differential cell counts were determined for each aliquot. Bovine serum albumin (BSA) with dimethylsulfoxide (DMSO) was added to half of the aliquots, and fetal bovine serum (FBS) with DMSO was added to the other half. One half of the aliquots containing BSA or FBS were frozen at -20 degrees C, and the other half were frozen at - 80 degrees C. After 3, 7, or 10 days, samples were thawed and total cell counts, viability, and differential cell counts were assessed.

RESULTS

Slide quality and total cell counts did not vary significantly according to freezing duration, temperature, or medium when compared to nonfrozen control samples. With FBS at -80 degrees C, cell viability did not vary significantly between control samples and freezing for 3, 7, and 10 days (59% vs 54%, 59% vs 54%, and 58% vs 54%, respectively; p > 0.05), whereas every other condition showed a significant decrease. Freezing did not affect the eosinophil percentage significantly.

CONCLUSION

Freezing of IS samples in FBS with DMSO at - 80 degrees C allows adequate preservation of IS specimens. Samples can be kept for at least 10 days in those conditions without significantly altering total cell counts, viability, and eosinophil percentage.