Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

Abstract

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 280 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 5 0.54-0.87; all P < 0.0001, and R 5 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 280 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. V C 2013 International Clinical Cytometry Society

A novel agent SL-401 induces anti-myeloma activity by targeting plasmacytoid dendritic cells, osteoclastogenesis and cancer stem-like cells

“…In this context, previous studies demonstrated that IL-3 R is restricted to certain myeloid progenitors, mature monocytes, eosinophils, basophils, mast cells and pDCs. 36 In our study, we found no significant effect of SL-401 on the viability of monocytes, B cells or total PBMCs. Although SL-401 would target normal pDCs, adverse immune effects or immunosuppression has not been observed in the clinical studies 14,15 to date, likely due to the restoration of normal pDCs post-SL-401 treatment.…”

Sputum basophils are increased in eosinophilic asthma compared with non‐eosinophilic asthma phenotypes

“…Whilst earlier studies were limited by the use of light microscopy and the small number of basophils in the airways, recent flow cytometric methods have considerably improved the ability to investigate rare cell populations in sputum . The aim of this study was to compare sputum basophil numbers between adult asthmatics and healthy controls and among asthma inflammatory subtypes.…”

“…Asthma (n=26) was identified on the basis of a doctor's diagnosis and current wheeze and, in Newcastle, was confirmed by demonstrated airway hyperreactivity to hypertonic saline. All participants provided written informed consent and successfully underwent spirometry, skin prick testing and sputum induction using hypertonic saline as described previously . Seven participants with inadequately controlled asthma at initial assessment (who were also amenable to asthma medication changes) had their treatment changed according to National Asthma Education and Prevention Program (NAEPP) Expert Panel Report 3 (EPR 3) guidelines and were re‐assessed 4‐6 weeks after their first visit to determine whether an increase in the prescribed ICS dose affected sputum basophil frequencies.…”

“…Sputum samples were processed according to a well‐characterized protocol, with staining and assessment of sputum leucocyte proportions by light microscopy to identify asthma inflammatory phenotypes (Eosinophilic asthma (EA) was identified on the basis of >2% eosinophils, and non‐ EA (NEA) (<2% eosinophils) was stratified into NA and PGA on the basis of 61% neutrophils). A fraction of sputum cells were prepared for flow cytometry as described previously . Briefly, samples were washed in Dulbecco's phosphate‐buffered saline (DPBS), incubated with live/dead fixable blue dye (Molecular Probes, Eugene, OR, USA) on ice for 15 minutes, washed again (in FACS buffer; DPBS with 10 mmol/L ethylenediaminetetraacetic acid (EDTA) (Sigma‐Aldrich, St Louis, MO, USA), 0.01% sodium azide (Sigma‐Aldrich) and 2% foetal calf serum (FCS; SAFC Biosciences, Lenexa, KS, USA) and stained for 30 minutes on ice with antibody panels containing anti‐CD45‐APC‐Cy7, anti‐CD14‐APC, anti‐CD16‐Alexa 700, anti‐CD123‐PE, anti‐FcER1‐FITC and anti‐HLA‐DR‐PerCP (all BD Biosciences, San Jose, CA, USA; titrated to optimal concentrations).…”

Similar activation state of neutrophils in sputum of asthma patients irrespective of sputum eosinophilia

“…These inflammatory phenotypes are identified by microscopically evaluating the percentages of neutrophils and eosinophils in sputum or by transcriptomic profiling of whole sputum samples [2,5,6]. In addition to microscopic evaluation, cells obtained by sputum induction can be analysed by flow cytometry [7][8][9][10][11]. Sputum granulocytes analysed by this technique have been shown to display an activated phenotype, with up-regulation of markers for activation and degranulation on either neutrophils [11], eosinophils [12] or both [13].…”