Responsivity and Reproducibility of Sputum Inflammatory Biomarkers During COPD Exacerbation and Stable Phases – A Pilot Study

Figures & data

Figure 1 Sputum induction protocol.

Notes: If the decrease in FEV₁ is >20%, the sputum induction is stopped. If the decrease in FEV₁ is 10 to 20% of the postsalbutamol inhalation FEV₁, patients receive 200 µg salbutamol. After 10 min FEV₁ is measured again. If FEV₁ is <90% of baseline FEV₁, the SI is stopped. After each step, the patients are asked to cough up sputum. As far as feasible, patients are encouraged to complete all of the steps of the entire protocol. ^aFEV₁ is measured.

Abbreviations: FEV₁, forced expiratory volume in 1 second; L, liters.

Table 1 Baseline Clinical and Demographic Characteristics

Number of patients	17
Sex (m/f)	13/4
History of exacerbations nb (number in last 12 months)
0	7	2 missing
1	6
≥2	2
	Median	25–75 percentile
Age (years)	70.0	69.0–71.0
Pack years smoking	39.0	29.0–58.0
BMI	28.6	24.9–31.8
Exacerbation, visit 1:
FEV1 (%pred)	56.0	46.0–66.0
FEV1/FVC (%)	46.0	43.0–51.0
Stable phase, visit 2
Days after exacerbation visit	55.0	42.0–73.0
FEV1 (%pred)	58.0	49.0–70.0
FEV1/FVC (%)	48.0	44.0–54.0
Stable phase, visit 3
Days after stable phase visit 1	7.0	7.0–10.0
FEV1 (%pred)	58.0	52.0–67.0
FEV1/FVC (%)	47.0	44.0–56.0
Sputum induction protocol (Cautious/Regular)	8/9

Notes: Data are presented as median (interquartile range: 25th−75th percentile).

Abbreviations: FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; pred, predicted.

Table 2 Sputum Inflammatory Cytokine Markers

Cytokine	Visit 1 Exacerbation	Visit 2 Stable State	Visit 3 Stable State	Responsivity: v1 and v2		Reproducibility: v2 and v3
				p-value (Δ V1–V2)	CV	ICC (95%CI)	Rho
IL-8
Protein (pg/mL)^a	6.01± 0.95	5.52 ± 0.80	5.49 ± 0.57	0.076	2.0	0.44 (−0.10; 0.78)	0.38
mRNA-Δ Ct values	−6.29 ± 2.54	−5.16 ± 0.77	−5.61 ± 2.68	0.11	2.1	0.34 (−0.47; 0.83)	0.57
IL-6
Protein (pg/mL)^a	5.88 ± 1.77	3.99 ± 2.32	4.59 ± 1.26	0.012*	1.4	0.11 (−0.23; 0.74)	0.41
mRNA-Δ Ct values	1.17 ± 3.20	3.17 ± 2.11	2.81 ± 3.0	0.16	2.0	0.51 (−1.6; 0.88)	0.52
CCL2
Protein (pg/mL)^a	3.67 ± 1.01	4.09 ± 1.19	4.01 ± 1.33	0.19	2.8	0.39 (−0.16; 0.75)	0.38
mRNA-Δ Ct values	−3.66 ± 1.90	−3.91 ± 1.42	−4.30 ± 1.14	0.63	6.4	0.69 (0.007; 0.93)	0.38
CCL4
Protein (pg/mL)	27.4 (6.4, 94.2)	0.0 (0.0, 37.8)	0.0 (0.0, 21.6)	0.013*	NA	NA	0.28
mRNA-Δ Ct values	−3.02 ± 3.10	−1.49 ± 2.21	−2.30 ± 2.59	0.22	2.4	0.29 (−0.27; 0.78)	0.38
TNF-α
Protein (pg/mL)^a	0.8 (0.0, 23.9)	0.0 (0.0, 0.0)	0.0 (0.0, 0.0}	0.053	NA	NA	0.31
mRNA-Δ Ct values	−1.89 ± 3.04	−1.12 ± 2.62	−2.28 ± 2.45	0.44	3.9	0.73 (0.16; 0.94)	0.76
LTB4 (pg/mL)^a	5.63 ± 1.55	6.25 ± 1.60	6.16 ± 1.32	0.22	3.0	0.59 (0.11; 0.85)	0.69

Notes: Data are presented as mean ±standard deviation or median (interquartile range: 25th−75th percentile); matched V1-V2 RNA level: N=10, matched V2-V3 RNA level: N=8, matched V1-V2 protein level: N=15, matched V2-V3 protein RNA level: N=14; lower Ct-values correspond with higher mRNA expression. Ct-values are normalized for 18S ribosomal RNA expression. ^aLog transformed; *p-value <0.05 (statistical tests performed on log-transformed parameters).

Abbreviations: v1, visit 1; v2, visit 2; v3, visit 3; CV, coefficient of variation; ICC, Intraclass correlation coefficient; CI, confidence interval; pg/mL, picogram per milliliter; Δ, delta; Ct, cycle threshold; mRNA, messenger RNA; LTB4, leukotriene-B4; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-8, interleukin-8; CCL2, CC-chemokine ligand 2; CCL4, CC-chemokine ligand 4; NA, not available due to non-normal distribution, see methods section; rho, Spearman correlation coefficient.

Table 3 Sputum Inflammatory Cells

	Visit 1 Exacerbation	Visit 2 Stable State	Visit 3 Stable State	Responsivity: v1 and v2		Reproducibility: v2 and v3
				p-value (Δ V1–V2)	CV	ICC (95%-CI)	Rho
Total cells Abs. count/mL^a	15.1± 1.5	14.9± 1.2	15.4± 1.0	0.65	6.72	−0.22 (−1.01; 0.64)	0.0
Neutrophils
%	13.9 (11.1, 23.9)	18.0 (3.6, 31.8)	24.0 (16.1, 32.9)	0.54	NA	NA	0.57
Abs. count/mL^a	17.8 ± 1.6	17.3 ± 1.9	18.3± 1.3	0.51	4.58	−0.42 (−1.1; 0.51)	0.07
Eosinophils
%	4.1 (1.0, 9.8)	4.0 (3.0, 8.4)	6.5 (3.0, 19.1)	1.00	NA	NA	0.43
Abs. count/mL^a	16.3 (15.2, 16.9)	16.6 (15.2, 17.6)	16.9 (16.5–18.2)	0.32	NA	NA	0.43
Macrophages
%	75.2 (59.4, 85.0)	70.6 (64.3, 87.0)	64.0 (54.3, 70.4)	0.92	NA	NA	0.68
Abs. count/mL^a	19.3 ±1.6	19.1 ±1.3	19.5 ± 1.1	0.57	5.35	0.14 (−0.84; 0.79)	0.25
Lymphocytes
%	0.00 (0.00, 0.10)	0.00 (0.00, 0.69)	0.00 (0.00, 0.06)	0.14	NA	NA	−0.39
Abs. count/mL^a	−0.7 (−0.7, 9.3)	−0.7 (−0.7, 14.0)	−2.3(−2.3–5.2)	0.40	NA	NA	−0.39

Notes: Data are presented as mean ±standard deviation or median (interquartile range: 25th−75th percentile); matched V1–V2 samples: N=10; matched V2–V3 samples: N=7. ^aLog transformed.

Abbreviations: CV, coefficient of variation; ICC, intraclass correlation coefficient; CI, confidence interval; v1, visit 1; v2, visit 2; v3, visit 3; Abs. count/mL, absolute counts per milliliter; NA, not available due to non-normal distribution, see methods section; rho, Spearman correlation coefficient.