Appendix (for Online Data Supplement)



Patient characteristics

Altogether 146 infants (boys 67%) referred for investigation of lower respiratory tract symptoms underwent clinical assessment, including the measurement of FENOand whole body plethysmography. 48/146 infants underwent bronchoscopy as part of their clinical evaluation. In 36/48 infants, an endobronchial biopsy specimen suitable for assessment of inflammatory cells was obtained. Their characteristics have been shown in Table 1.

Table 1. Characteristics of the infants in the study.



Male (%)81

Age (months)*12.5 (3.4-25.9)

Height (cm)*77.1 (61.7-88.1)

Weight (kg)*10.6 (7.4-14.7)

Gestational age (weeks)*40.0 (36.0-42.0)


Parental asthma (%)36

Parental smoking (%)31

Main respiratory symptom

cough (%)39

wheeze (%)56

dyspnoe (%)6

Symptom duration (months)*6.0 (2.0-16.0)

Lung function

sGaw (z-scores)-2.1 (-4.6-1.7)

FRC (z-scores)0.5 (-2.0-3.3)


*median (range); #atopic eczema or skin prick test positivity


The infants were sedated with chloral hydrate (50-100 mg/kg) after which specific airway conductance (sGaw) and functional residual capacity (FRC) were measured. For the measurement of FENO, exhaled air was collected with a face mask placed over infants' mouth and nose, during tidal breathing (FENO,TB). The infants inspired room air, and expired, via a separate expiratory valve in the mask, at least 20 breaths into a Mylar sampling bag. No NO filter was used but ambient NO was monitored during the tests and observed to be low (<10 ppb). FENO,TB was analyzed offline with a chemiluminescence analyzer (CLD 77, Eco Physics, Duernten, Switzerland).

According to routine clinical assessment at the Hospital for Children and Adolescents and Skin and Allergy Hospital, Univesrity of Helsinki, the infants were evaluated with bronchoscopy to exclude structural airway abnormalities. Endobronchial biopsy was performed for research purposes. The study was approved by the local ethics committee, and written, informed consent was obtained from the parents.

Biopsies were processed, and plastic sections (1 µm) stained with toluidine blue. Ultrathin sections containing areas of epithelium, RBM, and subepithelium were cut for analysis of inflammatory cells. The numbers of subepithelial inflammatory cells (eosinophils, neutrophils, mast cells, plasma cells, and lymphomononuclear cells), identified by their ultrastructure, were assessed in ultrathin sections of infant biopsies using electron microscopy. As there are no reference data in normal healthy infants, we used an arbitrary classification based on the median count of each cell line (<50th percentile=low; 50th percentile=high).

Nonparametric tests were used for analysis: Mann-Whitney U for the comparison of NO levels between infants with high and low counts of cells, and Spearman for correlation analyses. Median FENO,TB (range) values were reported.


The median FENO,TB was 15.7 ppb (range 3.0-68.7 ppb), infants with atopic eczema showing slightly higher values (26.7 ppb) than those without eczema (16.6 ppb, p=0.07). The median (range) FENO,TBin infants classified according to the counts of inflammatory cells in the biopsy specimen, are presented in Table 2. The scatterplots of FENO,TB vs. inflammatory cell counts with correlation analyses have been presented in Figures 1-5.

Table 2. Median (range) FENO,TB in infants classified according to the counts of inflammatory cells in the endobronchial biopsies.


Cell typeLowHighp


Eosinophils15.7 (3.0-68.5)16.7 (4.1-68.7)0.71

Eosinophils*14.7 (3.0-27.2)26.7 (8.0-68.7)0.084

Plasma cells20.3 (3.0-68.5)11.3 (3.7-68.7)0.019

Neutrophils 19.6 (6.1-68.5)11.3 (3.0-68.7)0.046

Mast cells17.6 (3.0-68.7)14.3 (4.1-68.5)0.85


*only atopic infants included (n=15)