Projekte pro Jahr
Abstract
Rationale: Transbronchial lung cryobiopsy (TBLC) allows for bronchoscopic retrieval of
representative lung biopsies in the diagnostic work-up of ILD. We performed additional
transmission electron microscopy (TEM) of TBLC samples in patients undergoing routine ILD
evaluation. Methods: Patients undergoing TBLC additional to standard evaluation of newly
diagnosed or deteriorated fibrotic ILD gave written informed consent to TEM evaluation of one
TBLC specimen. Four to six biopsy specimens were routinely retrieved from two different lung
segments of one lung. For controls, unaffected subpleural lung tissue from lobectomy material of
patients undergoing lung resection for lung cancer was retrieved both by cryobiopsy through a
pleural incision and by excision without freezing. TEM specimens were fixed in a Karnovsky’s type
fixative, postfixed in OsO4, en-bloc stained with phosphotungstic acid (PTA), and embedded in
epoxy resin. Ultrathin sections were examined using a Zeiss LEO EM 910 microscope. Formalin-
fixed TBLC specimens served for routine histology and were additionally immunostained for
markers relevant to endothelial transformation (CD31, ASMA, LYVE-1 and FSP1) using a Leica
Bond RX stainer. Results: Eleven patients (9 men, 2 women, median age 71 years) provided
appropriate alveolar lung tissue for TEM analysis, which enabled good fine structural preservation.
Freezing artifacts (mitochondrial swelling, dilatation of intra- and extracellular spaces) occurred in
greatly standardized form. The main structural TEM findings concerned endothelial
micromorphology, with presence of endoluminal pseudopodia-like protrusions and inner surface
defects. This was extensively found in four (36%; stage 3), moderately in three (27%; stage 2), and
could not be detected in four patients (36%; stage 1). Comparison of frozen and not frozen controls
excluded freezing-induced causes. Patients with extensive vs. moderate vs. no TEM endothelial
abnormalities were younger (mean age 67 vs. 75 vs. 73 years), presented with a higher fraction of
clinical and serological autoimmune features (100% vs. 33% vs. 25%), higher broncho-alveolar
lavage lymphocyte fraction (mean 15% vs. 7% vs. 6%), and had lower forced vital capacity/diffusion
capacity for carbon monoxide (mean 63/48% vs. 83/54% vs. 83/54% predicted).
Immunohistochemical analysis of endothelial cells and adjacent perivascular tissue revealed no
abnormalities in CD31 and ASMA expression but increased LYVE1 and FSP1 reactive cells in the
endothelial layer of vessels and alveolar walls. Conclusions: Application of TEM on TBLC samples
in fibrotic ILD patients is feasible and identifies a subset of patients with ultrastructural endothelial
impairment which is likely to have implications for the clinical phenotype.
representative lung biopsies in the diagnostic work-up of ILD. We performed additional
transmission electron microscopy (TEM) of TBLC samples in patients undergoing routine ILD
evaluation. Methods: Patients undergoing TBLC additional to standard evaluation of newly
diagnosed or deteriorated fibrotic ILD gave written informed consent to TEM evaluation of one
TBLC specimen. Four to six biopsy specimens were routinely retrieved from two different lung
segments of one lung. For controls, unaffected subpleural lung tissue from lobectomy material of
patients undergoing lung resection for lung cancer was retrieved both by cryobiopsy through a
pleural incision and by excision without freezing. TEM specimens were fixed in a Karnovsky’s type
fixative, postfixed in OsO4, en-bloc stained with phosphotungstic acid (PTA), and embedded in
epoxy resin. Ultrathin sections were examined using a Zeiss LEO EM 910 microscope. Formalin-
fixed TBLC specimens served for routine histology and were additionally immunostained for
markers relevant to endothelial transformation (CD31, ASMA, LYVE-1 and FSP1) using a Leica
Bond RX stainer. Results: Eleven patients (9 men, 2 women, median age 71 years) provided
appropriate alveolar lung tissue for TEM analysis, which enabled good fine structural preservation.
Freezing artifacts (mitochondrial swelling, dilatation of intra- and extracellular spaces) occurred in
greatly standardized form. The main structural TEM findings concerned endothelial
micromorphology, with presence of endoluminal pseudopodia-like protrusions and inner surface
defects. This was extensively found in four (36%; stage 3), moderately in three (27%; stage 2), and
could not be detected in four patients (36%; stage 1). Comparison of frozen and not frozen controls
excluded freezing-induced causes. Patients with extensive vs. moderate vs. no TEM endothelial
abnormalities were younger (mean age 67 vs. 75 vs. 73 years), presented with a higher fraction of
clinical and serological autoimmune features (100% vs. 33% vs. 25%), higher broncho-alveolar
lavage lymphocyte fraction (mean 15% vs. 7% vs. 6%), and had lower forced vital capacity/diffusion
capacity for carbon monoxide (mean 63/48% vs. 83/54% vs. 83/54% predicted).
Immunohistochemical analysis of endothelial cells and adjacent perivascular tissue revealed no
abnormalities in CD31 and ASMA expression but increased LYVE1 and FSP1 reactive cells in the
endothelial layer of vessels and alveolar walls. Conclusions: Application of TEM on TBLC samples
in fibrotic ILD patients is feasible and identifies a subset of patients with ultrastructural endothelial
impairment which is likely to have implications for the clinical phenotype.
Originalsprache | Englisch |
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Seiten | A3262 |
DOIs | |
Publikationsstatus | Veröffentlicht - 2024 |
Veranstaltung | American Thoracic Society International Conference - San Diego, California, USA/Vereinigte Staaten Dauer: 17 Mai 2024 → 22 Mai 2024 |
Konferenz
Konferenz | American Thoracic Society International Conference |
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Land/Gebiet | USA/Vereinigte Staaten |
Ort | San Diego, California |
Zeitraum | 17/05/24 → 22/05/24 |
Systematik der Wissenschaftszweige 2012
- 106 Biologie
Projekte
- 1 Abgeschlossen
-
EMILDS: Pilotstudie Elektronenmikroskopie bei fibrosierenden interstitiellen Lungenerkrankungen und Sarkoidose
Stoiber, W. (Projektmitarbeiter/in) & Obermayer, A. (Projektleitung)
1/01/24 → 1/01/25
Projekt: Forschung