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Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry
Collin R Brooks1, Christine J van Dalen, Ian F Hermans
1Centre for Public Health Research, Massey University Wellington Campus, Wellington, New Zealand. c.r.brooks@massey.ac.nz
Insights
Flow cytometry (FCM) offers a reproducible method for analyzing airway inflammation via induced sputum leukocyte counts. While cryostorage impacts neutrophils, FCM remains a viable, objective alternative to traditional microscopy for differential cell counts.
Area of Science:
- Pulmonary Medicine
- Immunology
- Cell Biology
Background:
- Airway inflammation assessment traditionally uses sputum induction and differential cell counts (DCC) via light microscopy.
- Light microscopy-based DCC is limited by inter-observer variability and poor reproducibility.
- Developing objective methods for sputum analysis is crucial for accurate inflammation assessment.
Purpose of the Study:
- To develop and validate flow cytometry (FCM) as an objective method for quantifying leukocyte populations in induced sputum.
- To compare FCM with traditional light microscopy-based DCC for accuracy and reproducibility.
- To evaluate the impact of cryostorage on leukocyte populations in induced sputum.
Main Methods:
- Induced sputum samples from 41 adults (23 with asthma) were analyzed using both DCC and FCM.
- FCM utilized CD45, CD14, and CD16 antibodies to identify major leukocyte subsets.
- The effect of cryostorage at -80°C on 15 samples was assessed, alongside cell viability analysis.
Main Results:
- FCM and DCC showed significant correlations for both fresh and cryopreserved samples (R = 0.54–0.87).
- Cryostorage led to significant neutrophil loss (median 30.5% to 17.4%; P < 0.0001).
- Lymphocytes exhibited higher viability than granulocytes or macrophages; no significant differences were found between asthmatics and non-asthmatics.
Conclusions:
- Flow cytometry (FCM) is a suitable and objective method for assessing leukocyte populations in induced sputum.
- Cryostorage at -80°C is feasible but may negatively impact neutrophil counts, though correlations remain strong.
- Significant variations in cell viability highlight the potential necessity of viability dyes in FCM analysis.
Background:
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).
Methods:
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.
Results:
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.
Conclusions:
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.
