Optimising bronchoalveolar lavage: lessons from alpha-1 antitrypsin deficiency

Abstract

Background: Bronchoalveolar lavage (BAL) is essential in determining the efficacy of novel therapies in alpha-1 antitrypsin deficiency (AATD). These require initial proof-of-concept demonstration that treatment administration increases alpha-1 antitrypsin (AAT) levels and/or anti-neutrophil elastase inhibitory capacity (ANEC) in the lung. Early-phase studies often encounter high interindividual variability of BAL results, primarily stemming from the inherent dilution characteristics of returned BAL fluid. A BAL protocol that minimises this variability is needed for reliable comparison of biochemical endpoints in the lung.

Methods: The study population included 21 severe AATD (ZZ), 22 moderate AATD (MZ) and 23 non-AATD (MM) individuals, further categorised as healthy, unobstructed current smokers or patients with chronic obstructive pulmonary disease (COPD). An additional six ZZ individuals were receiving intravenous alpha-1 augmentation therapy. We compared common BAL correction methods-albumin, total protein and epithelial lining fluid (ELF) volume measured by urea-when reporting early-phase biochemical endpoints, AAT and ANEC.

Results: BAL performed with a paediatric bronchoscope (PB) improved alveolar sampling compared with a traditional adult bronchoscope. Both uncorrected and ELF-corrected BAL demonstrated high interindividual variability regardless of lung health status. BAL total protein correction minimised interindividual variability, producing significant differences in AAT and ANEC between all genotypes, the strongest relationship with plasma AAT levels (r²=0.83), greatest inter-lobar concordance in AAT levels (r²=0.76) and strong correlation between BAL AAT and ANEC (r²=0.88).

Conclusions: By capitalising on the marked consistency in AAT levels between AAT genotypes, and the close relationship between plasma and lung AAT levels, we demonstrate reliable alveolar sampling that aligns closely with plasma.

Keywords: Alpha1 Antitrypsin Deficiency; Bronchoscopy; COPD Pathology.

Figure 1. (A) Comparison of BAL return in healthy and COPD individuals regardless of AAT genotype (mean 55% vs 34%, p<0.0001), N=59 (table 1: healthy 22, COPD 37). (B) Comparison of BAL return in healthy and COPD (C) individuals by AAT genotype, N=59. (C) Correlation between FEV1 (% predicted) and BAL return (%) (N=59, Spearman r²=0.34, p<0.0001). AAT, alpha-1 antitrypsin; BAL, bronchoalveolar lavage; FEV1, forced expiratory volume in 1 s.

Figure 2. (A) BAL albumin concentrations in healthy, smoking (S) and chronic obstructive pulmonary disease (C) individuals for each AAT genotype. (B) BAL epithelial lining fluid (ELF) volumes recovered. (C) BAL total protein concentrations. Mean and SEM displayed. Comparisons made with analysis of variance. (D) Correlation between ELF AAT and BAL AAT corrected for albumin (Spearman r²=0.57, p<0.0001), and ELF AAT correlated with BAL AAT corrected for total protein (Spearman r²=0.79, p<0.0001). AAT, alpha-1 antitrypsin; BAL, bronchoalveolar lavage.

Figure 3. (A) Plasma AAT levels in non-AATD (MM), moderate AATD (MZ), severe AATD (ZZ) and ZZ AATD 48 hours after IV-AAT infusion (Aug), divided into healthy, smoking (S) and chronic obstructive pulmonary disease (C) groups. (B) Uncorrected BAL AAT levels. (C) ELF AAT levels. (D) Protein-corrected BAL AAT levels. All panels display PB BAL results in the same 72 individuals (table 1), unless otherwise specified. Individual values and mean displayed. Comparisons made with analysis of variance. AATD, alpha-1 antitrypsin deficiency; AAT, alpha-1 antitrypsin; BAL, bronchoalveolar lavage; ELF, epithelial lining fluid; PB, paediatric bronchoscope.

Figure 4. (A) Correlation between plasma AAT levels and uncorrected BAL AAT levels (Spearman r²=0.72, p<0.0001). (B) Correlation between plasma AAT levels and ELF AAT levels (Spearman r²=0.67, p<0.0001). (C) Correlation between plasma AAT levels and protein-corrected BAL AAT levels (Spearman r²=0.83, p<0.0001). All panels display PB BAL results in the same 72 individuals (table 1). AAT, alpha-1 antitrypsin; BAL, bronchoalveolar lavage; ELF, epithelial lining fluid; PB, paediatric bronchoscope.

Figure 5. (A) Correlation between ELF AAT levels in the upper and lower lobes in 23 paired samples (Spearman r²=0.41, p=0.001; Wilcoxon, p=0.15). (B) Correlation between protein-corrected BAL AAT levels in the upper and lower lobes in 23 paired samples (Spearman r²=0.76, p<0.0001; Wilcoxon, p=0.96). (C) Comparison of ELF-corrected BAL AAT levels between genotypes and health states in upper lobe (black dots) and lower lobe (grey dots) samples. (D) Comparison of protein-corrected BAL AAT levels between genotypes and health states in upper lobe (black dots) and lower lobe (grey dots) samples. AAT, alpha-1 antitrypsin; BAL, bronchoalveolar lavage; ELF, epithelial lining fluid.

Figure 6. (A) BAL AAT levels in healthy, smoking (S) and chronic obstructive pulmonary disease (C) patients for each AAT genotype. (B) BAL anti-neutrophil elastase capacity (ANEC). Comparisons made with analysis of variance. (C) Correlation between BAL AAT corrected for protein and BAL ANEC corrected for protein (Spearman, r²=0.88). All panels display PB BAL results from the same 72 individuals (table 1). AAT, alpha-1 antitrypsin; BAL, bronchoalveolar lavage; PB, paediatric bronchoscope.