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On-line supplement
METHODS
Patients
Subjects with CF had the diagnoses made on either a positive sweat test or detection of
2 CF disease causing mutations [1]; included patients with PCD were diagnosed on
conventional criteria[2] .
Indications for bronchoscopy
All fibreoptic bronchoscopies (FOB) were carried out for clinical indications. The
investigation is routinely performed in our centre shortly after a diagnosis of CF is made
in order to establish microbiological status and to allow treatment for pathogens where
present; this group of patients made up part of the CF cohort. The remainder of the CF
group and the PCD and bronchiectasis patients were undergoing the procedure because
of unexplained decline in respiratory status or clinical symptoms and, in the majority, an
inability to expectorate sputum for culture. Healthy controls were bronchoscoped for
other reasons such as upper airway examination or a history of haemoptysis.
Consent
Written informed consent was obtained from parents/guardians of children undergoing
clinically indicated FOB. Specific consent (and assent where appropriate) was obtained
to retain samples surplus to requirement, and obtain additional samples for research.
Bronchoscopy Protocol
The procedure was carried out under general anaesthetic as is usual practice within the
centre and the specific anaesthetic technique was determined by the consultant
anaesthetist. Most children have anaesthetic induction by inhaled sevofluorane via face
mask, with the remaining children receiving intravenous propofol. Anaesthetic
maintenance is with sevofluorene and intravenous propofol if needed. The airway was
most commonly maintained by facemask and manual chin lift. Laryngeal mask airway
(LMA) and endotracheal tube were used if deemed appropriate by the anaesthetic
consultant.
2.8 internal diameter Olympus BF-XP40, 3.6 mm BF-3C20 or 3C40, 4.0 mm BF-MP60 and
4.6mm BF-P2OD bronchoscopes were used, the choice being dictated by the size of the
child. An initial inspection of upper and lower airways was made prior to samples being
obtained. Where possible, no suctioning was carried out in the upper airway to prevent
contamination of samples with upper airway flora.
BALF collection and processing
BALF samples were obtained by instilling 3 aliquots of 1ml/kg (maximum aliquot volume
of 40ml) 0.9% saline at room temperature to the right middle lobe or most affected
lobe(s). The aliquots were pooled. 1ml aliquots were sent to clinical laboratories for
microbiology ,processed in accordance with CF trust guidelines [3] , and cytology where
cell differential was performed. Cellular differential was performed following
cytospinning for 3 minutes at 200 g, air drying then fixation with methanol and May-
Grünwald-Geimsa staining. The research aliquot was put immediately on to ice and used
for research purposes. A 50 μl aliquot of whole BALF was mixed 1:1 with trypan blue
(Sigma-Aldrich, USA) and a total cell count performed using a dual chamber Neubauer
haemocytometer (Assisten, Sondheim, Germany). The remaining BALF was centrifuged
at 4˚C, 2000 g for 10 minutes and the supernatant stored in aliquots at -80˚C. As per
current research governance stipulations, samples were coded and individual aliquots
item tracked.
Blood samples
Venepuncture was clinically indicated and an additional aliquot of ≤3 ml of blood was
taken for research (total volume never greater than 1 ml/kg). Blood samples were
obtained through venepuncture whilst under general anaesthetic. These samples were
centrifuged at 4˚C, 2200 g for 10 minutes and the serum carefully pipetted and stored in
aliquots of between 125 and 500μl at -80˚C.
Measurements
25(OH)D2 Vitamin D was measured in serum. Assays were performed by the
biochemistry department at Royal Brompton and Harefield NHS Foundation Trust. The
samples were analysed using mass spectrometry with high pressure liquid
chromatography (HPLC).
HBD-2 HBD-2 was measured in BALF samples using ELISA (Phoenix
pharmaceuticals, USA). This was performed as per manufacturers’ instructions and
detects HBD-2 from 7.8 – 500 pg/ml [4]. Samples were diluted 1:1 with assay buffer
making the detectable range 15.6 pg/ml – 1000 pg/ml. 14 samples were measured in
duplicate and paired values very similar (CoV median (IQR) 4% (0.3-10%). The remainder
were run in singlicate.
LL-37 LL-37 was measured in BALF using ELISA (Hyocult biotech). Samples were
undiluted and the ELISA performed as per manufacturer’s instructions hyocult [5].
Samples were initially diluted, but serial dilution experiments revealed that dilution
effect was not linear and therefore all reported results were performed on neat
samples. Samples could not be run induplicate due to resources available. The assay
allowed measurement over the range 0.1 to 100 ng/ml. Two samples fell outside the
range of the kit and so the lower limit of detection, 0.1 ng/ml, was used for these
samples.
Cytokines
Meso scale discovery (MSD) was used for measurements of cytokines in the BALF. IL-1β,
IL-2, IL-6, IL-8, IL-10, IL-12, IFN-γ, TNF-α, GM-CSF and IL-17a were measured using a
combination of single and multiplex cytokine assays (Meso Scale Discovery, Rockville).
Samples were run in singlicate. These cytokines were chosen due to their actions of
regulation of inflammation in the airway [6].
In order to measure the cytokines, an antibody is coated onto a well, or for the
multiplex assays, a range of antibodies are coated in a specific pattern. The samples are
prepared by the addition of a solution containing detection antibodies (anti-IL-2, anti-IL-
8 etc.) each of which is labelled with an electrochemiluminescent compound. During
incubation periods, binding occurs and the final solutions are used for the readings. The
MSD instrument measures the quantity of emitted light which provides a quantative
measurement of the desired compound.
BALF samples were processed as per the assay protocol with a few minor changes, as
the protocol was not written for BALF. 12 μl 10% BSA solution was added to each of the
samples (108μl) to achieve a concentration of 1% BSA within each sample. The
standards were made up in 1% BAS and not the diluent as stated in the protocol to allow
for increased protein content within BALF.
The upper limit of detection for all cytokines was 2500pg/ml. The lower limit of
detection was determined by the individual assay run and varied from 0.013 to
4.86pg/ml. Samples were run in singlicate due to financial resources available
Viral PCR
The numbers of the group with viral PCR performed was smaller because the clinical
laboratory changed its protocol during the study period and previously viral analysis was
performed using immunofluorescence technique; which has been proven insensitive
and so these data have not been analysed.
Clinical data collection
Clinical data were collected including patient demographics, height, weight and clinical
symptoms at the time of the bronchoscopy, and spirometry results and microbiology
surveillance cough swabs/sputum samples over a 3 year period.
Statistical analysis
Mann Whitney for 2 groups, Kruskal-Wallis with Dunn’s correction for multiple groups,
logistic regression for binary outcomes and Chi squared test was used for comparison of
categorical data. SPSS v21 (IBM Corporation, New York) and Graphpad prism v6
(Graphpad software, San Diego) were used.
Definition of vitamin D deficiency:
Original cut-off values [7]
Insufficient
<50 nmol/L
Relatively sufficient≥50 nmol/L
Alternative 1[7]
Insufficient< 75 nmol/L
Sufficient≥75 nmol/L
Alternative 2[8]
Insufficient
<50 nmol/L
Relatively sufficient50–74 nmol/L
Sufficient
≥75 nmol/L
Alternative 3 [9-11]
Deficient<25 nmol/L
Insufficient25–74 nmol/L
Sufficient≥75 nmol/L
Alternative 4 Deficient
<25 nmol/L
Insufficient
25–49 nmol/L
Relatively sufficient50–74 nmol/L
Sufficient
≥75 nmol/L
Table 1: Post hoc analyses were performed using different cut-off values for vitamin D sufficiency and insufficiency. Irrespective of which cut-off value was used, no difference was seen between sufficient and insufficient patients.
RESULTS
Vitamin D level and age
The median ages were not different across the 3 groups although the proportion of
children <1 year of age was significantly higher in the CF group (p<0.01)(Table 2)
reflecting the practice of routine FOB in newly diagnosed infants with CF in our centre;
the 6 healthy controls were also older. For the patients with CF, vitamin D correlated
inversely with age (r=-0.35, p=0.001) and therefore multiple regression was undertaken
where relevant. Given this finding, post-hoc analyses were performed excluding all
children under 2 years of age, and again including only the children 2 years of age and
above, with no alteration to outcomes. Multivariate analyses accounted for this age
effect.
TABLES AND FIGURES
Table 2 CF patients with bacteria isolated in their BALF had higher BALF total cell count, neutrophil count, neutrophil differential, blood neutrophils, and higher BALF LL-37, IL-10, IL-12p70, IL-1B, IL-2, IL-8 and TNF-a. Values shown are median (95% CI).
BALFculture +ve
BALFculture -ve
p-value P value <0.01
BALF absolute count (x 103/L) 1,035 (730 – 1,880) 368 (273– 525) < 0.0001 ****BALF neutrophil differential (%) 58 (42 – 71) 14 (8 – 23) < 0.0001 ****BALF neutrophil count ( x 103/L) 521 (209– 1142) 39 (23.2 – 81.6) < 0.0001 ****
Serum neutrophils ( x 109/L) 5.3 (4.3 – 6.3) 3.5 (2.7 – 4.3) < 0.0001 ****BALF LL-37 ng/ml 0.82 (0.49 – 1.13) 0.35 (0.30 – 0.43) < 0.0001 ****BALF HBD-2 (pg/ml) 166 (123 – 231) 119 (65.5 – 203.5) 0.16 nsTNF-α (pg/ml) 3.4 (1.53 – 9.11) 0.5 (0.15 – 0.80) < 0.0001 ****GM-CSF (pg/ml) 0.7 (0.26 – 1.90) 0.2 (0.13 – 0.81) 0.02 nsINF-γ (pg/ml) 0.1 (0.00 – 1.6) 0.0 (0.0 – 0.11) 0.02 nsIL-10 (pg/ml) 1.2 (0.43 – 2.17) 0.2 (0.11 – 0.42) < 0.0001 ****IL-12p70 (pg/ml) 0.6 (0.14 – 1.46) 0.06 (0.00 – 0.13) 0.0004 ***IL-1β (pg/ml) 43 (15.3 – 77.4) 4.2 (1.06 – 8.68) < 0.0001 ****IL-2 (pg/ml) 0.5 (0.11 – 1.48) 0.1 (0.02 – 0.47) 0.002 **IL-6 (pg/ml) 21 (9.30 – 31.3) 6.4 (4.21- 14.2) 0.04 nsIL-8 (pg/ml) 2,347 (1,307– 4,163) 442 (234– 6995) 0.0004 ***IL-17 (pg/ml) 0.07 (0.02 – 0.18) 0.03 (0.01 – 0.07) 0.09 ns
Table 3 CF patients with viral detection in their BALF by PCR did not differ from those with no viral detection in vitamin D or antimicrobial levels. Values shown are median (95% CI).
BALFViral PCR +ve
BALFViral PCR -ve
p-value P value <0.01
n 8 16Vitamin D (nmol/L) 69 (37 – 105) 47 (34 – 71) 0.13 nsBALF LL-37 ng/ml 0.71 (0.31 – 3.94) 0.42 (0.37 – 1.16) 0.8 nsBALF HBD-2 (pg/ml) 70.2 (15.6 – 456) 68.3 (15.6 – 176) 0.99 ns
OLS Figure 1:
The median (95% CI) serum 25(OH)D2 levels were 57 (52 – 66), 42 (26 – 51) and 57 (24 – 74) nmol/L in the CF, non-CF CSLD and the healthy control groups respectively. The non-CF CSLD group had a significantly lower vitamin D level than the CF group (p<0.01). Vitamin D levels of each of the 3 groups were examined according to their microbiological status; BAL culture positive (red circles) vs. BAL culture negative (black triangles) and no differences were seen within each group (see OLS for further details and viral data)
OLS Figure 2: CF patients who isolated bacterial or fungal organisms from their BALF had higher levels of LL37 than those with sterile BALF. Median (95% CI) 0.82 (0.49 – 1.13) vs. 0.35 (0.30 – 0.43 ng/ml (p<0.0001).
OLS figure 3: A significant (p < 0.001) positive correlation was seen between LL-37 and BALF absolute cell count, BALF neutrophil count and differential and inflammatory cytokines.
OLS Figure 4: In CF patients, BALF LL-37 correlated with cellular and soluble markers of inflammation
OLS Figure 5: In CF patients, there was no correlation between cellular and soluble markers of inflammation and BALF HBD-2
OLS figure 6: There was no relationship seen between FEV1 nor FVC and either of the antimicrobial peptides in CF patients. LL37 did not correlate with FEV1 (r = - 0.14, ns) or FVC (r = - 0.15, ns). Similarly HBD-2 did not correlate with FEV 1 (r = 0.01, ns) and FVC (r = - 0.07, ns).
OLS Figure 7: CF patients with positive bacterial culture of their BALF had a higher BALF absolute cell counts than CF patients in whom no bacteria was isolated (p<0.0001)(figure a). These patients also had higher BALF neutrophil count (b), neutrophil differential (c), serum neutrophils, IL-10 (d), IL-1β (e) ,TNF-α (p < 0.0001), IL-8 (p=0.0004)(figure f)
OLS Figure 8: Vitamin D levels of CF patients who isolated any organisms in their BALF were compared with CF patients with sterile BALF. No difference was seen between the culture negative and culture positive groups (median (CI); 63 (52-68) vs. 56 (47 – 63)
nmol/L, ns). This was also true for the non-CF CSLD group (median (CI); 43 (27 – 59) vs. 33 (21 – 60) nmol/L, ns).
OLS Figure 9: FEV1 values taken from the annual assessments (AA) closet to the time of the FOB (AA FOB), the AA 12 months prior to this (AA pre) and the AA 12 months later. (AA post). No correlation was seen between vitamin D and FEV1 at any of the time points.
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