Prospective, longitudinal study of plastic bronchitis cast pathology and responsiveness to tissue plasminogen activator

Abstract

Plastic bronchitis (PB) is a rare disease that often occurs in patients with congenital heart disease (CHD) who have undergone staged single-ventricle palliation. It is characterized by the formation of rubbery "casts" in the airways. PB treatment frequently includes inhaled tissue plasminogen activator (tPA). However, the efficacy of tPA to reduce cast burden is unknown. This is further complicated by our lack of knowledge of cast composition. We obtained spontaneously expectorated PB casts from children (n = 4) with CHD and one adult patient with idiopathic PB. Pathological assessment was made from paraffin-preserved samples. Casts were treated with phosphate-buffered saline (PBS) or tPA. Cast response to tPA was assessed by changes in cast weight and the production of fibrin D-dimer. Independent of dose, tPA reduced cast weight compared with PBS-treatment (P = 0.001) and increased D-dimer levels. Histological staining showed that PB casts from all patients were composed of fibrin and contained notable numbers of lymphocytes. Cast composition did not change over time. Collectively, these data support that in our PB patients, casts are composed of fibrin and are responsive to tPA treatment. This makes inhaled tPA a potentially viable option for symptomatic relief of PB while we work to unravel the complexity of PB pathogenesis.

Figure 1

Photograph of representative spontaneously expectorated cast from a patient with PB.

Figure 2

Representative light micrographs of a MSB stained section (10X), in which fibrin stains red (A); a hemotoxylin and eosin stained section (600X), which highlights cellular content (B); and mucicarmine (mucin) stained sections which show a cast where mucin (dark pink) is localized in the cast periphery (arrows) (C) and one with no remarkable mucin (D).

Figure 3

Percent cast weight reduction normalized to the ratio of the amount of added tPA to the dry weight of each cast sample (A). Each data point represents a section (n=40) from a whole cast. At least three sections from each of 11 cast samples were tested for tPA responsiveness using different amounts of tPA and PBS as a negative control. There was no evidence of a tPA dose response (p > 0.05 by ANOVA; also see discussion) so data were pooled and compared with PBS treatment (B). tPA treatment resulted in a greater reduction in cast weight compared with PBS (*p = 0.001 by unpaired Student’s t-test). Data are the mean (+SD) of 11 PBS-treated and 29 tPA-treated cast samples.

Figure 4

Changes in active tPA concentration over time in the presence of glu-plasminogen and cast (A). The initial rapid decline in tPA concentration is indicative of plasminogen activation that occurred to a similar extent at all tPA doses. During incubation of cast with PBS, tPA was detectable at all time points (B). Standard deviations were removed for clarity. Data are the mean of 4–10 cast samples for each amount of tPA and PBS. The lower limit of detection of our tPA assay was 0.04 ng/mL.

Figure 5

Amounts of active tPA varied regionally in PB casts from two patients, one who received inhaled tPA (A) within 24 h of cast expectoration and one who did not (B). Casts were sectioned into 1) proximal; 2) mid; and 3) distal sections and homogenized. The amount of tPA (μg) in each piece was normalized to protein content (μg) of the respective homogenate.

Figure 6

Fibrin D-dimer increased over time with tPA treatment providing additional evidence of the tPA responsiveness and fibrin content of PB casts in our patients. These data also corroborate the presence of tPA in cast since D-dimer was evident in samples treated with PBS. In the absence of cast, D-dimer was not detected in PBS or at any of the tPA doses (data not shown). Data are the mean of three cast samples at each time point for each amount of tPA. Standard deviations were removed for clarity. The lower limit of detection of our D-dimer assay was 3.1 ng/mL.