A fnding of raised neutrophils (>5%) and eosinophils (>2%) is characteristic but not diagnostic of IPF [16]; in a minority of patients, it is possible to fnd a mild BAL lymphocytosis, which seldom exceeds 25% of the differential cell count [17]. BAL in IPF should be considered in all patients with suspected infection, malignancy. In such cases, it might be diagnostic.

Various types of acute and subacute lung injury can cause severe reactive pneumocyte atypia: diffuse alveolar damage (of known or unknown cause), “explosive” organizing pneumonia, acute fbrinous and organizing pneumonia (AFOP). In BAL these cells may be detected and they present with marked cytomegaly, vacuolated cytoplasm, prominent nucleoli, frequent binucleation. Recently a peculiar aspect labeled as “Napoleon hat” sign has been described [18, 19]. BAL is also useful in the detection of disseminated epithelial neoplasms and lymphoproliferative disorders [19, 20].

In addition to changes in the number and proportion of cells, other fndings in the BALF analysis are useful in ILD diagnoses such as the presence of lipid-laden macrophages (in chronic microaspiration or lipoid pneumonia) [Fig. 30.1c] and mineral dust (in some pneumoconiosis) [Fig. 30.1b] (Table 30.3).

The 2022 guidelines from the American Thoracic Society/ European Respiratory Society on IPF suggest that BAL could be indicated for patients with a frst ILD diagnosis of apparently unknown cause with a CT pattern of probable usual interstitial pneumonia (UIP), indeterminate UIP, or an alternative diagnosis [21].

BAL may also have a prognostic value: some studies have reported that BAL fndings at the time of diagnosis re ect the severity of ILD and may predict the prognosis. Increased neutrophils with a low lymphocyte count suggest a poor prognosis in chronic/fbrosing HP and increased neutrophils have been reported to predict mortality in IPF [22]. Similarly, an increased neutrophil count in BAL may be associated with an unfavorable outcome in newly diagnosed patients with sarcoidosis and it may indicate the need for active treatment [23].

Despite the presence of some controversies, BAL should be regarded as a useful tool in the clinical management of ILD. However it should not be used as an isolated tool for making a diagnosis, but always be interpreted in the context of disease history, clinical, laboratory, and radiological fndings and it can become particularly important if the biopsy is not feasible.

New research frontiers could be related to BAL cell gene expression patterns (nucleic acid microarray analysis), correlation of gene analysis profles and proteomic analysis

with specifc diagnoses, detection of specifc infection via microbial oligonucleotide analysis, construction of diagnostic algorithms, and disease activity and management guidance.

Recent studies have found that cells secrete vesicles containing proteins, lipids and nucleic acids (including RNA, miRNA) involved in the signal transduction to neighboring and distant cells. Exosomes represent one type of such vesicles, and the exosome-associated microRNA has drawn a lot of attention because of its accessibility in body uids (plasma, serum, BAL uid) and of its potential role as a biomarker.

It has been demonstrated a decreased expression of miR-­ 30A-­5p in the BAL uid exosome of IPF patients compared to healthy subjects. The miR30a5p has been reported to suppress migration and invasion and have also antifbrotic role in liver fbrosis so that miR30a5p could be proposed as a potential biomarker for IPF [24].

The exosomes present in the BAL uid of sarcoidosis patients is characterized by the presence of miRNA (miR-­ 146a and miR-150), the expression of which correlates negatively with pulmonary function indices [25].

Transbronchial Biopsy (TBB)

Transbronchial lung biopsy (TBB) using exible forceps yields small biopsy samples, on the order of 1–3 mm in the greatest dimension, often with signifcant crush artifact [26]. The procedure itself is associated with a low complication rate: pneumothorax (2–10%), severe bleeding (less than 2% of cases). Other complications (including acute exacerbation of interstitial lung diseases or air embolism) are even rarer. TBB is a common diagnostic technique used to defne lesions that have a centrilobular, alveolar, and/or perilymphatic distribution [1]. The lung specimens obtained with these small forceps come from the centrilobular regions and the zone centered upon small airways, so that lung disorders centered around terminal bronchioles or along the lymphatic routes may be reached by these forceps.

Therefore it is diagnostic mainly in organizing pneumonia, sarcoidosis, chronic eosinophilic pneumonia, diffuse alveolar damage, organizing pneumonia (OP) [Fig. 30.2], subacute hypersensitivity pneumonitis, low-grade B cell lymphomas and carcinomatous lymphangitis.

On the other hand, TBB is rarely suffcient to establish the diagnosis with confdence in diseases characterized by heterogeneous histological patterns or in those with the main histological abnormalities located in the periphery of the secondary lobule (such as UIP). Barbescu et al. reported that

Fig. 30.2  Trans-bronchial biopsy of the lung showing myxoid fbroblastic plugs typical of organizing pneumonia (OP). (Source: Pathology Department, G.B. Morgagni - L. Pierantoni Hospital, Forlì, Italy)

TBB can allow the recognition of the hall marks of the UIP pattern [27]: when patchy fbrosis, honeycombing, and fbroblastic foci are recognized, its specifcity for the UIP pattern appears to be high.

However, TBB has a very low sensitivity and specifcity when other patterns, such as nonspecifc interstitial pneumonia (NSIP) and desquamative interstitial pneumonia (DIP) are identifed [28]. It should also be considered that the sensitivity of the procedure is very low because of frequent crash artifacts resulting in the diffcult interpretation of areas of patchy fbrosis.

Recently it has been suggested that with the use of a genomic-based machine trained to identify a specifc molecular signature identifed from RNA sequencing on tiny transbronchial lung biopsy samples, the sensitivity of the procedure increases signifcantly. In this prospective study, the classifer recognized UIP pattern in transbronchial lung biopsy samples with 90% specifcity and 62% sensitivity [29].

The 2018 guidelines from the American Thoracic Society/ European Respiratory Society on IPF make no recommendation for or against the use of conventional TBB in patients with newly detected ILD of apparently unknown cause, clinically suspected of having IPF and with an HRCT pattern of probable UIP, indeterminate, or suggesting an alternative diagnosis. For patients with newly detected ILD of apparently unknown cause, clinically suspected of having IPF and with an HRCT pattern of UIP, TBB is strongly not recommended [26].

Transbronchial Lung Cryobiopsy (TLCB)

Before the recognition of the multidisciplinary (MDT) diagnosis as the gold standard for ILD diagnosis, surgical lung biopsy (SLB) was considered the reference diagnostic procedure to obtain suffcient histological information to distinguish UIP patterns from other interstitial lung diseases [30]. SLB is associated to risky complications, and costs. Adverse events include chronic chest pain, observed in more than 50% of the cases and lasting for months, prolonged air leakage, and infections. The mortality rate, when performed in a elective setting is around 2%, increasing signifcantly in elderly, in patients witha clincial diagnosis of IPF or Collagen Vascular Disease, or in those with rapid progressive pulmonary function deterioration. Pneumothorax is usually not considered a complication because it occurs in 100% of cases but the patient’s discomfort related to the chest tube should be considered [31]. Balancing the risk/beneft ratio, surgical lung biopsy is obtained in <15% of ILD cases, and the indication of biopsy has to be carefully considered by the multidisciplinary team that plays the leading role in the diagnostic process of ILDs.

Transbronchial lung cryobiopsy (TLCB) is a promising and less invasive alternative to surgical lung biopsy (SLB) to diagnose interstitial lung diseases (Table 30.4) [32–35].

TLCB allows to obtain larger and higher quality lung tissue samples without the crushed artifacts seen with conventional transbronchial lung biopsy using exible forceps, but the specimens are usually smaller than those obtained by surgical lung biopsy (SLB) [36].

Samples obtained through cryoprobe are usually 40–50 mm2 in size and contain peripheral structures of the secondary pulmonary lobule (visceral pleura, interlobular septa) allowing to recognize morphologic patterns of ILDs with high confdence, especially UIP pattern [37]. The optimal lung biopsy specimen size in the diagnosis of ILDs has not been established; anecdotally pathologists suggest that adequate specimens should measure at least 5 mm in diameter [35]. Different studies document that a diagnostic morphologic pattern may be identifed in more than 80% of subjects with ILD through TBLC [38]. Casoni et al. demonstrated that pathologists can detect UIP patterns with high confdence in about half of the cases with a very good overall interobserver agreement and that the diagnostic

Table 30.4  Comparison of SLB and TBLC

Fig. 30.3  Trans-bronchial lung cryobiopsy (TLCB) showing diffuse alveolar wall thickening by uniform fbrosis with interstitial lymphocyte in ammatory cells infltration and Masson bodies flling bronchiolar lumina and alveolar ducts and spaces (organizing pneumonia with overlapping non-specifc interstitial pneumonia). (Source: Pathology Department, G.B. Morgagni - L. Pierantoni Hospital, Forlì, Italy)

yield is related to the size of specimens [38]. Histological diagnosis of the UIP pattern is feasible on TLCB: elements of the UIP pattern (patchy fbrosis, fbroblastic foci, honeycomb changes) can be identifed with high confdence in cryosamples and the inter-observer variability between expert pathologists for diagnosis of the UIP pattern on TLCB seems to be similar to that described in SLB [39, 40] [Fig. 30.3].

Studies comparing the diagnostic accuracy of TBLC and SLB in the same patients who underwent consecutively both procedures are limited. The COLDICE study (Cryobiopsy versus Open Lung biopsy in the Diagnosis of Interstitial lung disease allianCE) showed high levels of agreement between TBLC and surgical lung biopsy for both histopathological interpretation and MDT. The TBLC MDT diagnoses made with high confdence were particularly reliable, showing excellent concordance with SLB MDT diagnoses [41].

The TLCB technique has been standardized [37, 38, 42]. Intubation with dedicated oro-tracheal tubes or rigid tubes are the preferred approach. C arch uoroscopy is strongly suggested and Fogarty baloon or bronchial blocjkers are considered mandatory in preventing or controlling major bleeding. Four to six biopsies are usually taken [43].

Ravaglia et al. showed that the strategy of performing two biopsies obtained from two different segments within the same lobe may be associated with an increased diagnostic yield [44]. In a larger series of 699 patients, Ravaglia and colleagues, showed that the diagnostic yield was signifcantly in uenced by the number of samples taken and the sampling strategy, improving dramatically when ≥2 samples were performed (instead of only one) and when biopsy was

obtained in two different sites (yields did not differ whether sites were represented by different segments of the same lobe or segments coming from different lobes) [44].

TBLC is a safe procedure, the main complications described in the literature are represented by pneumothorax, prolonged air leak, and transient respiratory failure. Acute exacerbation of the underlying ILD has been reported in less than 0.3 per cent of cases In a recent meta-analysis it was reported that the risk of pneumothorax can be in uenced by procedure-related factors, like the type of sedation/airway control: a higher proportion of pneumothorax occurred among intubated patients undergoing the procedure under deep sedation with invasive jet ventilation compared to patients under sedation and spontaneous breathing. The risk of pneumothorax seems to be in uenced also by patient-­ related factors (such as radiological fbrotic score and UIP pattern), or procedure-related factors (such as distance from the pleura, size of the probe used, the skill level of the operator) [32].

Bleeding is the most life threatening complication of cryobiopsy.

In the Ravaglia and colleagues study moderate bleeding was reported in 7.6% of patients and severe bleeding (resolved with prolonged Fogarty balloon bronchial occlusion but requiring admission to intensive care unit and prolonged intubation for <6 h) in 0.7% of patients [45–47].

The risk of acute exacerbation needs to be evaluated before the procedure: the presence of new patchy ground glass areas on CT scan [45], increased dyspnea on exertion in the last month, and/or high levels of in ammatory or more specifc markers (KL-6) could be predictors of high acute exacerbation risk [48].

Currently, the absolute contraindications for TBLC are the presence of a bleeding diathesis, anticoagulant therapy, treatment with thienopyridines and new antiplatelet drugs, and thrombocytopenia with a platelet count of less than 50 × 109/L. Patients with clinical or radiological signs of pulmonary hypertension should have a preprocedural evaluation and if the estimated systolic pulmonary artery pressure on echocardiography is >50 mmHg, this represents a relative contraindication. Acute deterioration in respiratory status should be considered a relative contraindication, such as severely impairment of pulmonary function (diffusing capacity <35% or forced vital capacity <50%) even if some reports are present in literature, indicating that TBLC is safe in this group of patients. Anecdotal data suggest that complications are more frequent when the pulmonary function is severely impaired. In a large cohort of 699 patients who underwent TLCB to investigate ILD, pneumothorax incidence was signifcative higher when FVC was <50% but it was not in u- enced by DLCO value. So that FVC <50% should be considered as a relative contraindication to transbronchial cryobiopsy. Signifcant hypoxemia, defned as PaO2 <55–

60 mmHg on room air or while receiving 2 L/min of nasal oxygen, has also been considered a contraindication [49]. Also a high body mass index (BMI) can result in failure of the procedure may be related to desaturation during the procedure [50]. Recently the European respiratory Society presented clinical guidelines on transbronchial cryobiopsy that will help clinicans to insert thia technique in the diagnostic work-up of patients with ILD [51] and transbronchial lung cryobiopsy is suggested as a valid alternative to SLB in the recent IPF/progressive pulmonary fbrosis guidelines [52].

Endobronchial Ultrasonographic

Transbronchial Needle Aspiration (EBUS-­

TBNA) and Endo-esophageal

Ultrasonographic Fine Needle Aspiration

(EUS-FNA)

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is often required to evaluate enlarged mediastinal and hilar lymph nodes, frequently associated with ILDs [53–55].

In sarcoidosis, EBUS and EUS-guided TBNA have a great diagnostic value, especially when combined with endobronchial and transbronchial biopsies [56, 57]. The diagnosis of lymphoproliferative disorders may be done with this approach but the use of smaller cryoprobes (1.1 mm) is advised [58].

EUS may have the advantage of allowing the sampling of sub-diaphragmatic organs, contributing to the diagnosis of lymphomatoid granulomatosis or other kinds of lymphoproliferative disorders involving the liver, spleen, or even adrenal glands.

Fig. 30.4  EBUS-TBNA performed on a mediastinal lymph node in a patient with nodular sclerosis (cellular phase) Hodgkin’s lymphoma (black arrow shows Reed Sternberg cell). (Source: Pathology Department, G.B. Morgagni - L. Pierantoni Hospital, Forlì, Italy)

Clinical Vignette

We report the case of a 45-year-old female evaluated in our clinic complaining of progressive dyspnea, dry cough, and weight loss in the last 3 weeks. The patient reported substantial clinical wellness up to 3 weeks ago with no signs or symptoms suggestive of collagen vascular disease.

She was a never smoker and her past medical history was notable for gastroesophageal re ux, and allergic asthma (diagnosed 5 years before).

Asthma was well controlled with medical treatment but in the last 2 years, the patient reported three respiratory exacerbations requiring oral steroids and antibiotics treatment. She also reported the presence of increased eosinophils levels in blood tests performed 1 year before (eosinophils 2900/mmc).

At the point of our presentation, she was on treatment with uticasone and salmeterol, omeprazole, and antiacids. On physical examination, sporadic wheezing was detected.

The patient’s laboratory investigations revealed increased WBC level (tot 20,100/mmc, Eosinophils 6750/ mmc42.9%), increased PCR 31 mg/L, negative proteinuria, and Bence Jones test, IgE normal level, negative IgG to Aspergillus. Autoimmunity work-up (including ANA, ENA, ANCA, anti-CCP) was negative.

Pulmonary function tests, performed at the presentation, showed a mild obstructive pattern (FVC 119% FEV1 93% Tiffenau 0.67), normal walking test with no signifcative desaturation, a mild decreased DLCO (69%), and normal blood gas values.

CT scan showed ground glass areas in the dorsal segment of the right upper lobe and in the apical segment of the left and right inferior lobes (Figs. 30.5 and 30.6).

Bronchoscopy with BAL and cryobiopsies (in two segments of the right upper lobe) was carried out. The differential cell count on BAL uid showed eosinophilia [Fig. 30.6] (total cells 735,000, neutrophils 16%, eosinophils 73%, lymphocytes 8%, macrophages 3%) while microbiology tests were negative (included SARS COV2). Cryobiopsies samples showed diagnosis was compatible with chronic pulmonary eosinophilia [Fig. 30.7]. Tests for mutation of JAK2 and FLIP1 were negative.

The diagnosis after multidisciplinary discussion was CEP.

The patient started treatment with methylprednisolone 40 mg intravenously for 3 days then oral prednisone 25 mg/die (0.5 mg/kg/die) for 4 weeks and