Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

Airway inflammation is commonly assessed by sputum induction followed by a differential cell count (DCC) using light microscopy. This method is prone to intercounter variability and poor reproducibility. We aimed to develop a more objective method using flow cytometry (FCM).

Fifty-six sputum inductions were conducted in 41 adults (23 asthmatics). Sputum was processed, a cytospin prepared for DCC, and the remainder immunolabeled for FCM using CD45, CD14, and CD16-specific antibodies to distinguish major leukocyte populations. Aliquots of 15 samples were frozen at -80°C to assess the effects of cryostorage. DCC and FCM were compared, and viability of individual cell populations was determined by FCM.

FCM and DCC, and fresh and frozen samples, were significantly correlated, R = 0.54-0.87; all P < 0.0001, and R = 0.57 to 1; P < 0.005, respectively. There was a significant neutrophil loss after cryostorage (from median 30.5-17.4% of total leukocytes; P < 0.0001). Cell viability was higher for lymphocytes compared to granulocytes or macrophages (P < 0.001). With the exception of the expected higher levels of eosinophils (P < 0.005), no significant difference in cell differentials or viability was observed between asthmatics and nonasthmatics using either DCC or FCM.

FCM is a suitable means of assessing leukocyte populations in induced sputum. Sample storage at -80°C prior to FCM is feasible, but may be detrimental to neutrophils, although good correlations were still observed between fresh and frozen samples. Large differences in viability were found between individual cell populations suggesting that viability dye use may be necessary.