Educational Material

ERS Annual Congress Amsterdam 26–30 September 2015

EDUCATIONAL MATERIAL

Educational Skills Workshop 20, 22, 24 Respiratory sampling techniques

Thank you for viewing this document. We would like to remind you that this material is the property of the author. It is provided to you by the ERS for your personal use only, as submitted by the author.

Tuesday, 29 September 2015 ESW20 08:00–10:20 ESW22 10:40–13:00 ESW24 14:30–16:50

Room L002 RAI You can access an electronic copy of these educational materials here:

http://www.ers-education.org/2015ew20

To access the educational materials on your tablet or smartphone please find below a list of apps to access, annotate, store and share pdf documents. iPhone / iPad

Adobe Reader - FREE With the Adobe Reader app you can highlight, strikethrough, underline, draw (freehand), comment (sticky notes) and add text to pdf documents using the typewriter tool. It can also be used to fill out forms and electronically sign documents. http://bit.ly/1sTSxn3

UPAD Lite - FREE UPAD Lite is an advanced note-taking application with annotation features. You can handwrite notes, highlight text, add sticky notes and reference images and export any type of document as a PDF or PNG file by email or to cloud services. http://bit.ly/1mQ1j0K

Noteability - $4.99 Noteability uses CloudServices to import and automatically backup your PDF files and allows you to annotate and organise them (incl. special features such as adding a video file). On iPad, you can bookmark pages of a note, filter a PDF by annotated pages, or search your note for a keyword. http://bit.ly/TCrNad

Android

Adobe Reader - FREE The Android version of Adobe Reader lets you view, annotate, comment, fill out, electronically sign and share documents. It has all of the same features as the iOS app like freehand drawing, highlighting, underlining, etc. http://bit.ly/1deKmcL iAnnotate PDF - FREE You can open multiple PDFs using tabs, highlight the text and make comments via handwriting or typewriter tools. iAnnotate PDF also supports Box OneCloud, which allows you to import and export files directly from/to Box. http://bit.ly/1p2SV00 ez PDF Reader - $3.99 With the ez PDF reader you can add text in text boxes and sticky notes; highlight, underline, or strikethrough texts or add freehand drawings. Add memo & append images, change colour / thickness, resize and move them around as you like. http://bit.ly/1kdxZfT

Educational Skills Workshop 20, 22, 24 Respiratory sampling techniques

AIMS: Sampling the airways is a useful way of accessing “the core” of lung diseases. Since the different techniques that are used to sample the airways can yield interesting immunological, inflammatory, and clinical data, this workshop aims to provide practical skills for both clinical and research purposes. The workshop does not require previous experience in this field. The workstations will be organised to be interactive and to provide all the required skills needed to perform the techniques.

TARGET AUDIENCE: Researchers, respiratory physicians interested in biological research, biologists, pulmonologists, pathologists, technicians.

CHAIRS: C. M. Kaehler (Innsbruck, Austria), P.A. Schnabel (Homburg/Saar, Germany), A. Spanevello (Tradate, Italy)

FACILITATORS: U. Costabel (Essen, Germany) , C. Kaehler (Innsbruck, Austria), P. Pignatti (Pavia, Italy), H. Popper (Graz, Austria) , E. Schmutz (Heidelberg, Germany) , A. Spanevello (Tradate, Italy) , E. Thunnissen (Amsterdam, Netherlands), M. Wiebel (Heidelberg, Germany)

WORKSHOP PROGRAMME PAGE

Four 30-minute workstations run simultaneously on the following topics:

Workstation 1 – Clinical cytology TBNA and FNA – Cytology and R.O.S.E. rapid on-site evaluation 5

Workstation 2 – Histology 11

Workstation 3 – Sputum collection Summary 94 Information of best practice 126

Workstation 4 – Analysis of BAL fluids 128

Additional resources 224

Faculty disclosures 225

Faculty contact information 226

Answers to evaluation questions 227

Regular physical activity improves quality of life and ﬁ tness in healthy individuals and people living with In partnership with: lung conditions.

www.healthylungsforlife.org

Farbe/colour: PANTONE 288 CV

ERS/ELF ER Journal 210x280 ad_v2 AW.indd 1 29/07/2015 16:22 TBNA and FNA – Cytology and R.O.S.E. rapid on-site evaluation

Dr Matthias Wiebel Thoraxklinik at University of Heidelberg Amalienstrasse 5 69126 Heidelberg GERMANY [email protected]

AIMS

Demonstration of practical aspects of ROSE with creating fast stained smears and highlighting favoring aspects and problems SUMMARY Mediastinal structures like lymph nodes (LN) or tumor need to be assessed in the diagnostic evaluation and staging of benign and malignant diseases. Invasive procedure like mediastinoscopy or thoracotomy can be replace by transbronchial needle aspiration (TBNA) or fine needle aspiration (FNA). The introduction of ultrasound guided fine needle aspiration using specially developed bronchoscopes and gastroscopes have greatly improved the localization of mediastinal lymph nodes and the yield of fine needle cell aspiration (FNA, TBNA).

The amount of cells collected in a sample depends on:

1. The size of LN, the puncture site and directabilty of the needle, 2. The underlying pathology (tumor, necrosis, vascularization), 3. The number of needle movements inside the LN, 4. The number of punctures (repeated sampling) in one LN position.

The result will also be influenced by the bronchoscopist:

1. Personal experience in EBUS and/or EUS, 2. Optimization of the endoscopic situation (appropriate sedation, application to the specific procedure), 3. Evaluation of the specimen retrieved (blood, clot, lymph liquid, necrosis, pus), 4. Ability to produce a good smear (mainly monolayer, no or little clots, no or little crushing artifacts due to the smearing process)

The further processing includes:

Smearing of specimens on glass slides and staining (May-Grünwald-Romanowsky / Giemsa with airdrying fixation; Papanicolau with ethanol spray fixation and secondary glass covering of the slide), and / or collecting the aspirated material in a container with a fixation medium (alcohol, formalin) for cytoblock techniques.

Follows the cyto-pathological evaluation:

The time needed depends on the cell numbers displayed on the smears, the ability to detect the origin of cells (lymphatic, tumor, necrosis, inflammatory). The additional amount of blood “dilutes” the cells needed to produce a diagnostic statement. The time needed is also depending on the numbers of slides to be evaluated. All in all the inspection for adequate material will reach from seconds to a couple of minutes. In addition every slide needs to be marked for patient data and site of puncture. In cases with less preserved vitality of cells time for evaluation will extend up to 15 minutes.

5 The results will be available within hours to days after the invasive diagnostic procedure.

The report will quote for:

1. Appropriate material: cells numbers, smearing quality (monolayer, undamaged cells) or inappropriate material: blood, scarce cells, artifacts like crushing of cells, reduced vitality or necrosis of cells. 2. Pathologic cells identified: tumor cells or aggregate, epithelioid cells or aggregates, macrophages and materials phagotized, inflammatory cells. 3. Additional specific staining for tumor identification may follow on spare slides.

The report will summarize the findings including the patient history: no pathology detectable or epithelial malignancy with differentiation into small cell cancer, non-small cell cancer (possibly suggesting squamous cell cancer or adenocarcinoma, metastatic from other known malignancy), malignant lymphoma, or epithelioid cells suggestive of granulomatous disease.

ROSE

The standard way of TBNA/FNA will produce situations with non-sufficient material for diagnosis. A further/additional (repeat) diagnostic procedure will be necessary: this means a severe burden for the patient due to the invasive procedure, a loss of time to start with therapy, additional costs. The same is true for the medical system.

A possible solution is advocated with the rapid evaluation of material on site (ROSE) retrieved by FNA/TBNA: Using a fast staining method (one to three minutes) and a microscope in the endoscopy room with an experienced microscopist, slides can be evaluated as to sufficient cellularity (lymphocytes or /and other cells in sufficient amount). Eventually diagnosis may be given as well (tumor, granulomatosis etc.). The main aim will be to quote whether the material gained is sufficient for diagnosis or an additional puncture is needed.

Structures needed for ROSE:

1. a microscope 2. rapid staining dyes, 90-second process

a. air-dried smear. Romanowsky Wright-Giemsa stain (a combination of Wright and Giemsa stains), and the buffered Wright-Giemsa stain, (four steps: buffer, eosin Y, azure B, and methylene blue) (e.g. Diff Quick). Technician for primary evaluation will be sufficient. b. ethanol spray fixation of smear. Ultrafast Papanicolaou stain, achieves comparable visual clarity in significantly shorter time (use 4% formaldehyde in 65% ethanol or 90% ethanol fixative, Richard-Allan Hematoxylin-2 and Cyto-Stain. The presence of a cytopathologist is needed.

Optimization of ROSE results:

Even though ROSE is intended to be fast in producing results time is needed for a reliable statement: the materials have to be marked, the staining and microscopic evaluation need to be correct. Thus three to five minutes per single ROSE slide series are appropriate and should be respected by the endoscopist. An initial learning curb to get a standardized habitual process should be accepted by all involved.

6 In the recently published Guidelines of TBNA acquisition and preparation of specimens the following questions are quoted (citation in italics): Can Rose increase the diagnostic yield? No. Can Rose decrease the number of aspirations? No. But additional punctures may be helpful in the individual setting.

Can Rose decrease the procedure time? No. Can Rose decrease the number of additional procedures? Yes. Rose may help avoid an additional bronchoscopy.

Can Rose decrease the rate of complications? No. The procedure are mainly unchanged, but the time of the procedure may be prolonged.

Is ROSE more cost-effective? There is no significant evidence for or against cost. But the time needed by the cytopathologist or the technician may be important.

What is the concordance of ROSE with the final diagnosis? The concordance is reported to be high.

Is there an optimal staining method? No, but Diff Quick seems to be the most applied staining set.

Who needs to perform ROSE? Cytopathologists will be able to evaluate specimens for adequacy and put forward a probable or even definitive diagnosis. Technicians and seemingly trained pulmonologists may at least decide on sufficiency of specimens.

TBNA and FNA add importantly to the armamentarium of diagnostic procedures in pulmonology. The addition of Rose can improve and speed up the diagnostic process. The successful implementation of ROSE requires dedicated endoscopists and cytopathologists.

REFERENCES

1. E.H.F.M. van der Heijden et al. World Association for Bronchology and Interventional Pulmonology. Guideline for the acquisition and preparation of conventional and endobronchial ultrasound-guides transbronchial needle aspiration specimens for the diagnosis and molecular testing of patients with known or suspected lung cancer. Respiration 2014;88:500-517

TBNA droplet and smearing procedure

Slide smearing. Left: clot dispersion by dragging the material similar to dab smear in solid biopsy. Right: saving spilt over material

Left: Fast staining Hemacolor (like DiffQuick). Right: spray fixation for rapid Papanicolau staining

Smear slides: (left to right) almost empty; broad droplet smear slightly uneven with fine aggregates at the end; drag smear of droplet with diminishing thickness; droplet with local coagulation ring and thin smearing

Smear slides: left thick droplet thin smear, right coarse spreading

Micoscope workplace with electronic camera and screen

Microscope screenshot:left: mainly monolayer activated lymphocytes; middle left: monolayer anisokaryosis and moulding in SCLC; middle right: squamous cell cancer and necrosis; right: anisokaryosis and preserved cytoplasm

Smear slide: left: anthracosis with damages macrophages; middle lymphocytes and macrophages; right: epithelioid granuloma and lymphocytes Copyright for all microscopic images by Dr. M. Wiebel, Heidelberg, Germany

10 Respiratory sampling techniques

Prof. Helmut H. Popper Pathology Medical University Graz Auenbruggerplatz 25 8036 Graz AUSTRIA [email protected]

Dr Erik Thunnissen Pathologie VU University Medical Center De Boelelaan 1117 1081 HV Amsterdam NETHERLANDS [email protected]

AIMS

To understand:  different respiratory sampling techniques, and  interaction required with pathologists to obtain as much analysis as possible.

SUMMARY

Success in the cyto-histological diagnosis of lung cancer depends upon, amongst other things, the correct sampling and processing of tissue. Summarised by a working group.[1]

Sampling techniques

Bronchial washing can be taken before or after biopsy sampling. Recommended is to recover as much as possible washing fluid after instilling 20-30 ml of isotonic saline.

BAL increases the diagnostic yield in pulmonary peripheral lesions and is primarily used for diagnosing infections and interstitial lung diseases.

Bronchial brushing is very useful in endoscopically visible tumours. Care should be taken to avoid withdrawing the unsheathed brush through the working channel of the bronchoscope. The area of suspected malignancy should be brushed two or three times.

Sputum is not recommend sputum as suitable material for diagnosis of lung cancer, due to its low sensitivity. However, in patients, who are unable to have a bronchoscopy or other minimal invasive procedure and where only diagnosis of malignancy is needed, sputum analysis may be helpful. Repeated sputum sampling over several days increases the sensitivity. Shedding of tumor cells can also be provoked by inhalation of prewarmed isotonic sodium-chloride solution prior to sputum collection.

Transbronchial needle aspiration cytology is frequently used. The diagnostic yield of conventional (non/ultrasound guided) transbronchial needle aspiration (TBNA = without endobronchial ultrasound, synonymous “EBNAB”) is high for endoscopically visible bronchial abnormalities, suggesting neoplasia, particularly when the lesion is due to extrinsic compression, submucosal infiltration, or an exophytic mass.

Linear Endobronchial Ultrasound (EBUS)-TBNA has been shown to have a high-pooled sensitivity of 93% and specificity of 100% for the confirmation of malignancy.

Endoscopic ultrasound (EUS) is especially useful for sampling posterior mediastinal and paraoesophageal lymph nodes (stations 4L, 7,8 and 9). In addition the left adrenal can be accessed and identified in 97% of cases. Combined EBUS and EUS is as sensitive as surgery for staging the mediastinum in lung cancer.

Biopsies of endobronchial visible tumours have a yield of 75-95% for a histological diagnosis of malignancy, after taking at least three forceps biopsy samples.

In patients with solitary peripheral pulmonary nodules, the endoscopic diagnostic procedure is usually performed as transbronchial lung biopsy (TBBx) under fluoroscopic or stereologic guidance. However, for peripheral lesions transthoracic needle biopsy (core needle or needle aspiration) is frequently used, because of the larger sample size.

Rapid On Site Examination (ROSE) is a quick cytological examination for the presence of tumour or lymphoid cells by pathologist or trained person. Initially, ROSE was set up for conventional (non- ultrasound guided) TBNA, for confirming the representativity of the sample. However, with the aim of obtaining as much tumour material as possible to allow more biomarker testing, the original goal is redundant, and additional needle passes may be required to obtain further tissue for molecular testing. Video assisted thoracoscopic (VATS) or surgical open lung biopsies re used for diagnosis of interstitial lung diseases or lung tumors.

Resection (bi-lobectomy, pneumectomy) is usually performed as treatment of lung cancer. Each procedure involving cytology and histology has their own artifacts at microscopic level.

Preservation of tissue architecture can be achieved by either instillation of freezing medium diluted 1:2 by PBS (frozen section diagnostics), or formalin. Lung tissue can thus be expanded and artificial collapse can be counteracted.

Pathology

The best strategy for the handling of small samples in suspected lung cancer is always based on accurate and relevant clinical information 1) sample site(s), 2) Whether the clinical suspicion is on primary tumour / metastasis; 3) Question diagnosis? If appropriate: also predictive test (s)?; 4) Previous relevant surgical, oncological, radiotherapeutic treatment, if any; 5) Smoking history (never, ex (pack years, years since quitting), current smoker (pack years, years of smoking).

This information will be used by the pathologist to i) determine priorities of the diagnostic approach (see below), ii) determine how specific a diagnosis is required, iii) plan the necessary investigations and anticipate the use of IHC and molecular biological tests, and iv) prevent unnecessary usage of tissue on tests which are not required or irrelevant given the clinical picture. Thereby, preserving scarce tissue for the most important tests required in each particular case.

Cytology is a powerful tool in the diagnosis of lung cancer. The advantages of diagnostic cytology are that it is a minimally-invasive, simple procedure, helps in faster reporting, and it is relatively inexpensive. The accuracy of the cytological examination depends greatly on the quality of collection, preparation, staining and interpretation of the material. However due to small sample size possibility for predictive analysis is limited.

12 Many cases can be adequately diagnosed (identified and typed), within the limits imposed by the nature of the specimen, using standard morphological stains (H&E, mucin stain or cytological stain(s) of choice).

The first step is the identification of malignancy and in clinical suspicion of lung cancer, the positive identification of carcinoma, as opposed to alternatives: i) lymphoma, sarcoma or other malignancy, ii) where relevant, in situ, non invasive, pre-invasive or reactive changes (hyperplasia, dysplasia etc), iii) non-neoplastic pathological processes (inflammation, benign tumours, etc) and iv) artefacts which may mimic neoplasia.

If carcinoma is identified, it may be assumed in many centres that the default diagnosis is primary lung cancer, but an adequate clinical history is essential to indicate if there is any suspicion or possibility that the lung tumour under investigation could be metastatic disease. The pathologist must always be alert to the possibility of metastatic disease, however primary lung cancer has a wide range of appearances. In the absence of a suggestive history, metastatic disease may be suspected from the morphological appearance of the carcinoma. The unnecessary or inappropriate pursuit with immunohistochemistry (IHC) or exclusion of a diagnosis of extra-thoracic carcinoma, metastatic to lung can potentially waste a great deal of time, laboratory resources and valuable tissue, since many options are possible and specificity is notoriously low.

After accepting the tumour as primary lung carcinoma, the next step is to determine whether the tumour represents small cell lung cancer (SCLC) or non-SCLC (NSCLC). Up to around 40% of NSCLC cases, it will not be possible to ascribe a NSCLC subtype by morphology alone (NSCLC not otherwise specified, NSCLC-NOS). Using IHC the percentage of unclassifiable NSCLC, NOS is reduced to around 4%. Such cases should NOT be diagnosed as large cell carcinoma, but remain NSCLC-NOS. Proactive block cutting (based on clinical information) and reflex testing prevents wasting by avoiding recutting the block, but at the same time also holds a risk of losing quality of epitopes and DNA when not used shortly after cutting. Additional sections may be taken from a small sample tissue blocks at the time of initial sectioning for morphological diagnosis, in anticipation of IHC and predictive analysis.

REFERENCES

1. Thunnissen E, Kerr KM, Herth FJ, Lantuejoul S, Papotti M, Rintoul RC, Rossi G, Skov BG, Weynand B, Bubendorf L, Grunberg K, Johansson L, Lopez-Rios F, Ninane V, Olszewski W, Popper H, Jaume S, Schnabel P, Thiberville L, Laenger F. The challenge of NSCLC diagnosis and predictive analysis on small samples. Practical approach of a working group. Lung Cancer [Internet] 2011/12/06 ed. 2012; 76: 1–18 Available from: http://www.ncbi.nlm.nih.gov/pubmed/22138001.

EVALUATION

1. Rapid on site evaluation of during cytology sampling for lung cancer is performed with EBUS/ EUS /TTBA. What is appropriate for predictive analysis? a. When a node is negative continue sampling. b. When node is positive continue sampling same node. c. When node is negative go to next enlarged node. d. When node is positive go to next enlarged node.

13 2. If a transbronchial biopsy did not yield in diagnostic tissue in lung cancer or an interstitial disease, what would you do next? a. repeat TBA b. proceed to VATS. c. perform EBUS TBNA d. perform BAL

3. Can an UIP diagnosis we made on cryobiopsies? a. no b. yes c. yes, but a differential diagnosis of underlying diseases is most often not possible d. no, the material is not sufficient

4. On cytologic specimen the diagnosis of mucin negative NSCLC NOS was made. What should be done next? a. Immunohistochemistry for TTF1 and p40 b. Immunohistochemistry for p63 and cytokeratin 5/6 c. Immunohistochemsirty for Surfactant Apoprotein A and cytokeratin 7 d. Immunohistochemsirty for cytokeratin 7 and napsinA

5. Does it matter, if a lung tissue specimen is placed in a large container with formalin, or is any container suitable? a. does not matter b. does matter, but I don’t know why c. does matter, because of fixation artifacts induced by squeezing the tissue d. does matter, because the tissue can stay therein for many hours

6. Can molecular testing be done on bone biopsies? a. yes, bone metastasis is equal to any other metastasis specimen b. no, bone metastasis shows less genetic changes than other metastasis c. no, DNA is degraded by acidic solutions used for decalcification d. yes, by decalcification normal tissue is destroyed and cancer tissue remains

14 RESPIRATORY SAMPLING TECHNIQUES

Helmut Popper Erik Thunnissen

15 Conflict of interest disclosure Popper  I have no, real or perceived, direct or indirect conflicts of interest that relate to this presentation.  I have the following, real or perceived direct or indirect conflicts of interest that relate to this presentation: Affiliation / financial interest Nature of conflict / commercial company name

Tobacco-industry and tobacco corporate affiliate related NO conflict of interest

Grants/research support (to myself, my institution or Unrestricted research grants from department): Eli Lilly (IALT-BIO), Astra Zeneca, Pfizer, Hofman La Roche, Boehringer-Ingelheim Honoraria or consultation fees: Advisory board honoraria from Hofman La Roche, Eli Lilly, Boehringer-Ingelheim, Novartis, Pfizer Participation in a company sponsored bureau: NO

Stock shareholder: NO

Spouse/partner: NO

Other support or other potential conflict of interest: NO

This event is accredited for CME credits by EBAP and speakers are required to disclose their potential conflict of interest going back 3 years prior to this presentation. The intent of this disclosure is not to prevent a speaker with a conflict of interest (any significant financial relationship a speaker has with manufacturers or providers of any commercial products or services relevant to the talk) from making a presentation, but rather to provide listeners with information on which they can make their own judgment. It remains for audience members to determine whether the speaker’s interests or relationships may influence the presentation. Drug or device advertisement is strictly forbidden. 16 Conflict of interest disclosure Thunnissen  I have no, real or perceived, direct or indirect conflicts of interest that relate to this presentation.  I have the following, real or perceived direct or indirect conflicts of interest that relate to this presentation: Affiliation / financial interest Nature of conflict / commercial company name

Tobacco-industry and tobacco corporate affiliate related Na conflict of interest

Grants/research support (to myself, my institution or Na department):

Honoraria or consultation fees: Eli Lilly support meeting European experts 2010

Participation in a company sponsored bureau: Na

Stock shareholder: Na

Spouse/partner: Na

Other support or other potential conflict of interest: Na

AIMS

• Understand different sampling techniques

• Understand interaction required to obtain optimal diagnostic and predictive analysis

18 Five items with essential clinical information for pathology diagnosis

1 Sample site(s) 2 Whether the clinical suspicion is on primary tumour / metastasis 3 Question diagnosis? If appropriate: also predictive test (s)? (D+P?) 4 Previous relevant surgical, oncological, radiotherapeutic treatment, if any 5 Smoking history (never, ex (pack years, years since quitting), current smoker (pack years, years of smoking)

19 20 HISTOLOGY LUNG CANCER

• Small cell lung cancer (SCLC)

• Squamous cell carcinoma (SqCC)

• Adenocarcinoma (AdC)

21 NSCLC – NOT OTHERWISE SPECIFIED (NOS) Resection specimen Biopsy / Cytology

Large cell carcinoma 1%

NOS 34%

Travis et al. J Thorac Oncol 2011;6:244–285; Thunnissen et al Virchows Arch. 2012;461:629–638 22 HE HE

p63 PAS-D

NSCLC Favors TTF-1 adenocarcinoma

Thunnissen et al. Virchows Arch 2012;461:629–638 23 24 Adenocarcinoma ´NOS´ 25 NSCLC: ADENOCARCINOMA VS SQUAMOUS CELL CARCINOMA Histology P63/40 TTF1 Mucin Squamous cell carcinoma +++ – – Adenocarcinoma –/+ + – –/+ – + –/+ + + NOS –/+ – – +++ + + • 85–90% favour adenocarcinoma or squamous cell carcinoma • Remaining: – Bx NOS (not otherwise specified) – Rx large cell/adenosquamous carcinoma

26 NSCLC: H&E + IMMUNO TTF1, P63

Resection specimen Biopsy / Cytology

Large cell carcinoma 0,2% NOS 4%

27 WHAT IS AN ACCEPTABLE RATE FOR NSCLC NOT OTHERWISE SPECIFIED (NOS)? • A diagnosis of NSCLC-NOS should be given in <10% of cases. [This figure is achieved with the judicious use of immunohistochemistry in morphologically indeterminate cases]

• A recommended approach should include TTF-1 to predict adenocarcinoma. For predicting squamous cell carcinoma, p63 or p40 and CK5/6 testing are useful

Kerr et al. Ann Oncol 2014;25:1681–1690

28 MODEL QUANTITATION IMMUNOHISTOCHEMISTRY INDIRECT TYRAMIDE/POLYMER IHC VS. INDIRECT SABC IHC Fluorescence intensities of immunohisto- chemical versus direct labeling DIG Cy3 = indirect polymer IHC = indirect SABC IHC 20 * ++ * *

( Thousands) * Absorption

Fluor escence escence IFluor nt ensity ( AU) * 0 1 * 10 80 -

DNA concentrationSpot = t edepitope DNA concent concentration r at ion ( ng/ ul)

Semi Q: - - - +/- + ++ ++ Absorption Modified from Prinsen CF, et al. Appl Immunohistochem Mol Morphol. 2003 ;11:168-73. 29 Linear dynamic range = quantitative range, more signal enhancement: closer to black and white; positive/ negative

= indirect polymer IHC * = indirect SABC IHC ++ * *

* Absorption

* * -

Difference between positive and negative: factor 2 in concentration

Modified from Prinsen CF, et al. Appl Immunohistochem Mol Morphol. 2003 ;11:168-73. 30 MODEL QUANTITATION IMMUNOHISTOCHEMISTRY INDIRECT TYRAMIDE/POLYMER IHC VS. INDIRECT SABC IHC Linear dynamic range = quantitative range, More signal enhancement: closer to black and white; positive/ negative = indirect polymer IHC * = indirect SABC IHC ++ tyramide * *

t * Absorption

* * -

negative positive

Difference between positive and negative: factor 2 in concentration

Modified from Prinsen CF, et al. Appl Immunohistochem Mol Morphol. 2003 ;11:168-73. 31 IMMUNOHISTOCHEMISTRY (IHC) FOR ALK IN LUNG CANCER

• Two antibodies (5A4, D5F3) work equally well • Testing can be quick • IHC used for screening will detect most ALK positive cancers • Confirm ALK IHC+ cases with FISH

32 Marchetti et al. J Thorac Oncol 2013; 8:352–358; Kerr et al. Ann Oncol 2014;25:1681–1690; Thunnissen, et al. Virchows Arch. 2012;461:245-57 33 PATHOLOGIST PRACTICE

34 CELL AND TISSUE ACQUISITION

• Sputum • Biopsy from • Scopy ~ 1 mm – Bronchus – Peripheral – Lymph node small needle cytology • Puncture through chest wall – Pleural fluids – Lung trans-thoracic needle biopsy 1 cm x 0.8 mm

• Metastatic sites: lymph node 1 x 0.5 cm, brain, bone (decalcify EDTA)

• Autopsy 2 m

35 36 37 PATHOLOGY FORM

Where is material sampled?

Fixative, time start fixation?

Clinical info: - Tumor - Suspicious for primary/ metastasis - Predictive analysis?

38 DIAGNOSIS BY CYTOLOGY AND HISTOLOGY

39 TRANSBRONCHIAL BIOPSY

The yield is usually low The reason might be that there is often a peritumoral inflammatory or scarring, which prohibits the biopsy forceps to reach the tumor Other problems are insufficient tissue, tinny pieces from the periphery of the tumor

40 TRANSBRONCHIAL BIOPSY BAL Can be diagnostic in some diseases Usually TBX is not very useful in ILD unless some lesions are located in more central locations Follow patterns seen in HRCT: If there are nodular lesions TBX might sample diagnostic tissues – take a minimum of 4 biopsies

41 TRANSBRONCHIAL BIOPSY BAL Can be diagnostic in some diseases Usually TBX is not very useful in ILD unless some lesions are located in more central locations Follow patterns seen in HRCT: If there are nodular lesions TBX might sample diagnostic tissues – take a minimum of 4 biopsies

42 TRANSTHORACIC NEEDLE BIOPSY

43 TRANSTHORACIC NEEDLE BIOPSY

44 45 46 47 48 49 50 51 ANALYSIS BY RESECTION MARGIN FROZEN SECTIONS

➠

52 TUMOR CAN ALSO BE ANALYSED THIS WAY

53 Fixation Dehydration in alcohol Xylol Paraffin (56’C)

54 55 56 57 4 μm coupes

58 59 60 Automatic stainer for H&E

61 Inside automatic stainer

62 example

63 TISSUE MANAGEMENT

• Many doctor’s responsibility • Lung cancer ‘hidden’ organ • Small samples

64 PATHOLOGY: OLD FASHION

Pulmonologist: Clinical information Question: diagnosis?

Pathology: Cut many sections

65 NEW FASHION: TISSUE MANAGEMENT

Pulmonologist: Clinical information Questions: diagnosis, prediction “D+P”, if malignant

Pathology “D+P” code: specific handling in contrast to regular

D+P

NOT performing these additional cuts saves material... Adequate clinical information is essential

66 MORE ANALYSIS FROM ONE BIOPSY?

HE first D+P Diagnostic stain TTF1 Diagnostic stain mucin Diagnostic stain P63/p40 Predictive stain ALK IHC Predictive stain ALK FISH DNA isolation EGFR DNA isolation DNA isolation HE last

67 “1st”

“last”

68 TISSUE MANAGEMENT = INTERACTION BETWEEN PULMONOLOGIST/ONCOLOGIST/ SURGEON/RADIOLOGIST AND PATHOLOGIST

Sample collector: Pathology :

Sampling more (≥4) Distribute samples over biopsies/ tumor tissue >1 block

Clinical request for Careful initial cut diagnosis + prediction? Spare section for reflex Clinical suspicion of analysis metastases y/n? Focused diagnostic analysis

69 CANCER IN THE LUNG: PRIMARY VS METASTASES • Clinical information / PALGA* essential: • Primary pulmonary adenocarcinoma: Surfactant prot A, Napsin A, TTF1 • Metastases: • Colorectal: CK7, CK20, CDX2 • Prostate: PSA, PAP • Breast: ER, PR, GCDFP15, GATA3 • Germ cell: PLAP, AFPHcG, CD30, OCT3/4, Sox2, Sox17 • Melanocyte: Melan A, HMB45, Sox 10, MITF • Mesothelium: Calretinin, CK5/6, D2-40, WT-1 • Kidney: RCC, CD10, Pax2, Pax8 • Ovary: CA125, Pax5, Pax 8 • Pathological anatomy national automated archive – a nationwide histopathology and cytopathology network in the Netherlands

Nonaka et al. Diagnostic Histopathology 2010;16:581–592 70 CANCER IN THE LUNG: PRIMARY VS METASTASES • Clinical information / PALGA* essential: • Primary pulmonary adenocarcinoma: Surfactant prot A, Napsin A, TTF1 • Metastases: • Colorectal: CK7, CK20, CDX2 • Prostate: PSA, PAP • Breast: ER, PR, GCDFP15, GATA3 • Germ cell: PLAP, AFPHcG, CD30, OCT3/4, Sox2, Sox17 • Melanocyte: NOTMelan performing A, HMB45, Soxthese 10, stains MITF saves material.. • Mesothelium: Calretinin, CK5/6, D2-40, WT-1 Adequate clinical information is essential • Kidney: RCC, CD10, Pax2, Pax8 • Ovary: CA125, Pax5,FOR Pax optimal 8 TISSUE MANAGEMENT • Pathological anatomy national automated archive – a nationwide histopathology and cytopathology network in the Netherlands

Nonaka et al. Diagnostic Histopathology 2010;16:581–592 71 GUIDANCE ON TISSUE HANDLING: SPECIMEN PROCESSING • Standard fixation using 10% neutral buffered formalin (4% formaldehyde) is recommended [V, A]

• Fixation time should be no less than 6 h, and no greater than 48 h [IV,A] Record start of fixation time on clinical form

• Sections for biomarker testing should ideally be cut immediately before analysis [IV, A]

Kerr et al. Ann Oncol 2014;25:1681–1690 72 CYTOLOGY HANDLING

• Cytology samples (cell blocks, stained direct smears or liquid- based preparations) can be used reliably to detect EGFR mutations and ALK rearrangements [III, A]. At this time, a cell block is the most widely applicable cell source

Kerr et al. Ann Oncol 2014;25:1681–1690 73 STATION 7 ENLARGED PET POSITIVE

74 FNA/ EBUS/ EUS Cytology

ROSE

Rapid On Site Evaluation

Bulman et al. Am J Respir Crit Care Med 2012;185:606–611; Thunnissen et al. Lung cancer 2012,76:1–18 75 ROSE function changed:

Sample more from positive site Repeat more passes in tumor positive nodes

Make 2 smears (PAP MGG) cell block

Bulman et al. Am J Respir Crit Care Med 2012;185:606–611; Thunnissen et al. Lung cancer 2012;76:1–18 76 CYTOBLOCK PROTOCOL

• Thinprep™ Cytolyt solution (Hologic) • Postfix with formaldehyde 4 hrs RT • Centrifuge; pellet • Shandon™ Cytoblock™ (J Clin Pathol 1992;45:1122-1123;Thermo Scientific) • Transfer in alcohol 70% to cassette, use 2 sponges/ transfer casette in alcohol 70% with eosin/ VIP

Kulkarni et al. Diagn Cytopath 2009;37:86–90 77 Cytology Tumor cellularity

Sufficient for Diagnostic/ predictive testing ?

yes

78 CYTOLOGY ALK FISH PROTOCOL

• Online Resource 2 • Basel (Switzerland) protocol for FISH on cytological specimens (Papanicolaou-stained smears or cytospins); Vysis ALK break- apart assay • Day 1: • - Place the uncovered slide(s) for 2 minutes in 2xSSC at 73°C in a microwave • - Place slide(s) for 30 minutes in 0.5mg/lm Pepsin in 0.01N HCL at 37°C in a • microwave • - Place slide(s) for 5 minutes in formalin 1% / 100X MgCl2 (25ml 10% formalin, • 74ml PBS and 1ml 2M MgCl2) at room temperature (RT) • - Place slide(s) for 5 minutes in PBS at RT • - Place slide(s) for 1 minute in 70% ethanol at RT • - Place slide(s) for 1 minute in 80% ethanol at RT • - Place slide(s) for 1 minute in 100% ethanol at RT • - Let dry slide(s) for 10 minutes at RT • - Warm up heating plate to 45°C • - Place slide(s) for 2 minutes on the heating plate • - Apply 10μl ALK probe “ready to use” to the target area of the slide • - Cover area with coverslip (20x20mm) • - Seal slide(s) with rubber cement or parafilm • - Place slide(s) on the surface of the HYBriteTM (or ThermoBriteTM) • denaturation/hybridization system. Fill in empty slots with blank glass slides • - Close the lid of the HYBriteTM and start the following program: • o Co-denaturation: 8 minutes at 74°C o Hybridization: overnight at 37°C • Day 2: • - Warm up heating plate to 37°C • - Heat up water bath to 73°C together with a coplin jar containing 2xSSC/0.3% • NP40 wash solution • - Remove the rubber cement or parafilm and the coverslip from the slide(s) • - Place slide(s) in the 2x SSC/0.3% NP40 at 73°C. When all slides are in the coplin • jar incubate for 2 minutes • - Remove the slide(s) from the wash solution and place them in a coplin jar containing 2x SSC/0.1% NP40 and incubate the slides for 1 minute at RT • - Remove the slide(s) from the wash solution and place them vertically in the dark on a paper towel for drying 79 • - Apply 10μl DAPI I counterstain onto the target area and place a 24x32mm coverslip over the DAPI solution, avoiding air Experienced cytopathologist

• FISH is a robust technology applicable to almost all types and formats of cytological specimens, including conventional smears, cytospins, or liquid- based preparations. • Use adhesive-coated and positively-charged slides • FISH works equally well on unstained specimens as well as those stained with Papanicolaou, H&E or May-Grünwald-Giemsa • Precise relocation of tumour cells using an automated stage greatly facilitates FISH scoring and review, especially in cases with a low proportion of tumour cells on the slide.

80 CYTOLOGY ALK FISH PROTOCOL

• Online Resource 2 • Basel (Switzerland) protocol for FISH on cytological specimens (Papanicolaou-stained smears or cytospins); Vysis ALK break- apart assay • Day 1: • - Place the uncovered slide(s) for 2 minutes in 2xSSC at 73°C in a microwave • - Place slide(s) for 30 minutes in 0.5mg/lm Pepsin in 0.01N HCL at 37°C in a • microwave • - Place slide(s) for 5 minutes in formalin 1% / 100X MgCl2 (25ml 10% formalin, • 74ml PBS and 1ml 2M MgCl2) at room temperature (RT) • - Place slide(s) for 5 minutes in PBS at RT • - Place slide(s) forON 1 minute LINE in 70% ethanol SUPPLEMENT at RT • - Place slide(s) for 1 minute in 80% ethanol at RT • - Place slide(s) forThunnissen 1 minute in 100% ethanol et at RT al., Virch. Arch. 2012, ALK review • - Let dry slide(s) for 10 minutes at RT • - Warm up heating plate to 45°C • - Place slide(s) for 2 minutes on the heating plate • - Apply 10μl ALKResponsible probe “ready to use” to the authortarget area of the is slide Lukas Bubendorf: • - Cover area with coverslip (20x20mm) • - Seal slide(s) withexpert rubber cement on or parafilmcytology FISH • - Place slide(s) on the surface of the HYBriteTM (or ThermoBriteTM) • denaturation/hybridizatione-mail: system. Fill [email protected] in empty slots with blank glass slides • - Close the lid of the HYBriteTM and start the following program: • o Co-denaturation: 8 minutes at 74°C o Hybridization: overnight at 37°C • Day 2: • - Warm up heating plate to 37°C • - Heat up water bath to 73°C together with a coplin jar containing 2xSSC/0.3% • NP40 wash solution • - Remove the rubber cement or parafilm and the coverslip from the slide(s) • - Place slide(s) in the 2x SSC/0.3% NP40 at 73°C. When all slides are in the coplin • jar incubate for 2 minutes • - Remove the slide(s) from the wash solution and place them in a coplin jar containing 2x SSC/0.1% NP40 and incubate the slides for 1 minute at RT • - Remove the slide(s) from the wash solution and place them vertically in the dark on a paper towel for drying 81 • - Apply 10μl DAPI I counterstain onto the target area and place a 24x32mm coverslip over the DAPI solution, avoiding air INTERACTION BETWEEN PATHOLOGIST AND PULMONOLOGIST/ONCOLOGIST/SURGEON/RADIOLOGIST

• Pathologists knows diagnosis (D) and implications for personalised medicine (Prediction = P) agreement on action • Inform pathologist possible clinical reasons for metastases • Allows reflex testing when needed, due to previous communication, also reimbursements, timely molecular analysis • Turn around Time (TAT) EGFR and ALK 1 week. Max 10 working days. Extramural within 3 days, intramural within 24 hours

• Practice in Amsterdam Vumc: reflex for “D+P” NGS/EGFR, KRAS, BRAF, HER2, analysis and IHC ALK, ROS1 • Practice in Graz MUG: reflex for EGFR, EML4-ALK1 (IHC pre-test), ROS1 (IHC pre-test)

Lindeman et al. J Mol Diagn 2013;15:415–53; Kerr et al. Ann Oncol 2014; 9:1681–1690 82 • Requirement of the assay

• Sufficient amount of DNA for HRM/ seq

• 1~30% tumour cells

• NGS Miseq: 50 ng • Ion Torrent 10 ng

83 TTF1 p63 84 Lymphangitis carcinomatosa, Sufficient for diagnosis Not enough for prediction

TTF1 p63 85 • Sufficient DNA for HRM/ seq/ NGS • ~30% tumour cells

• Not enough tumour DNA • <(0.)1% tumour cells

86 • Sufficient DNA for HRM/ seq/ NGS • ~30% tumour cells

• Not enough tumour DNA • <1% tumour cells

• Cytology (block): same requirements

• Glass slide: once

87 “1st”

“last”

88 TISSUE MANAGEMENT = INTERACTION

Sample collector: Pathology :

Sampling more (≥4) Distribute samples over biopsies/ tumor tissue >1 block

Clinical request for Careful initial cut diagnosis + prediction? Spare section for reflex Clinical suspicion of analysis metastases y/n? Focused diagnostic analysis

89 BIOMARKER ANALYSIS

• The same pathologist should, if possible, review all available tumour material from the same patient including biopsies and cytology specimens to select the most suitable for biomarker analyses [IV, A]

• A pathologist should be involved in sample preparation for DNA extraction [V, A]

• Enrichment of samples by micro- or macrodissection to maximise tumour cell content before DNA extraction is recommended [III, A]

• Turn around time 10 days

Kerr et al. Ann Oncol 2014; 9:1681–1690; Lindeman et al. J Mol Diagn 2013;15:415–53; 90 FOR PRECISION MEDICINE: TUMOR SAMPLE

• Sample more tumor • Cervical lymph node: excision preferred over biopsy • More biopsies in different tubes/ blocks • Biopsy preferred over cytology • Cytology: more passes with `ROSE` • Smear work also for FISH, sequential testing, but limited options

91 CONCLUSIONS

• Interaction between doctors involved in the patient management is essential for optimal diagnostic and predictive analysis • Histology samples preferred, but cytology works well too (for limited number of predictive tests) • ROSE: more passes when tumor positive: cell block

92 WHAT TYPES OF TISSUES FOR RESEARCH

• EBUS FNA and Cytoblock can be used in tumor research • BAL useful for research in non-tumor diseases; also analysis of proteins from fluid • Transbronchial biopsy; low yield, less useful • Transthoracic biopsy useful in tumor research, but also in research on certain non-tumor conditions • VATS biopsy most useful; if preevaluated by frozen section, samples can be stored in liquid nitrogen and used fresh for all types of molecular analysis • Animal lung (rodents); most useful, but needs knowledge in anatomy and histology of mammals

93 Sputum collection

Dr Patrizia Pignatti Allergy and Immunology Unit Fondazione Salvatore Maugeri Via Salvatore Maugeri 10 27100 Pavia ITALY [email protected]

Prof. Antonio Spanevello Division of Pneumology Salvatore Maugeri Foundation IRCCS Via Roncaccio 16 21049 Tradate ITALY Department of Clinical and Experimental Medicine University of Insubria Via Guicciardini 9 21100 Varese ITALY [email protected]

AIMS

Among respiratory sampling techniques, sputum collection represents a non invasive way to assess airway inflammation. The aims of this workstation are:

 To highlight standardized procedures of sputum collection and processing  To evaluate the pros and cons of this methodology  To show difficulties and drawbacks  To discuss clinical utility.

SUMMARY

Sputum analysis is a useful method to evaluate airway inflammation in asthma, chronic obstructive pulmonary disease (COPD) and chronic cough both in research and in clinical practice. Induced sputum is needed when patients cannot spontaneously produce sputum allowing sampling of low airway secretions also in these subjects.

Sputum induction is performed through the inhalation of ultrasonically nebulised saline solution (isotonic or hypertonic) over different time periods. The patient is invited to cough and to collect secretions. The subject is asked to inhale 200 mg of salbutamol before induction, and forced expiratory volume in 1 s (FEV1) is monitored before and after each inhalation to either prevent or detect possible bronchoconstriction 1.

Sputum induction is a simple, safe and well-tolerated procedure even in patients with severe lung diseases and exacerbations. It is recommended that experienced staff applies standard operating procedures taking into consideration the degree of airway obstruction, use a modified protocol for subjects with severe airway obstruction, and assess lung function and symptoms during the procedure

94 Sputum induction is considered to be safe if the fall in FEV1 is within 5% of baseline after waiting 15 min. If a FEV1 fall > 20% compared to baseline occurs, the inhalation must be stopped. This adverse effect can affect 11% of asthmatics and patients with COPD 2.

After collection, sputum sample is processed within 2 hours according to a standardised method with mucolitic agents (dithiothreitol). Centrifugation is required to separate sputum cells from the fluid phase which is stored at -80°C for soluble mediator evaluation. If the soluble mediators are affected by mucolitic agents, sputum should be processed with phosphate buffer alone 3-4.

Sputum from a healthy subject is rich in macrophages and neutrophils and poor in eosinophils, lymphocytes and epithelial cells 5. Cut-off for sputum eosinophils varies from >2 or >3% according to different authors and ERS guidelines.

Asthma is characterised by sputum eosinophilia, which predicts a favourable response to corticosteroids. However, non-eosinopilic asthma accounts for 25–55% of steroid-naive asthmatics and is associated with a poor response to corticosteroids 6.

In up to 40% of subjects with chronic cough, a sputum eosinophil count >3% is shown. These subjects with cough, sputum eosinophilia and no lung function alterations receive the diagnosis of eosinophilic bronchitis, and have an objective response to corticosteroid treatment.

In COPD, neutrophils are usually increased and they are associated with reduced FEV1, suggesting that neutrophilic airway inflammation is functionally relevant. A cut-off for sputum neutrophilia should take into account age, since neutrophilis accumulate in the airways with aging. Sputum eosinophilia could be present in subjects with COPD and usually predicts a response to corticosteroid therapy 7. Sputum analysis can be useful also at exacerbation to classify the type of exacerbation 8.

This methodology has many pros: it is relatively non invasive, it’s quite rapid if performed by trained staff, it can be repeated during follow-up to monitor adhesion to therapy and change it if inflammation is not under control. Furthermore, from one single rapid and not expensive test you can get many data with different meanings: total cell count, cell viability, eosinophilic, neutrophilic or mixed inflammation.

The cons are: despite the induction procedure, few subjects cannot produce sputum; you cannot repeat the induction within 24 h in order to avoid neutrophilic inflammation due to the inhalation of the hypertonic saline solution, you need to have laboratory facility.

Many attempt to substitute sputum eosinophil data with surrogate markers of airway inflammation such as FeNO and blood eosinophils still did not reach the same sensitivity and specificity of sputum analysis 9. Furthermore, to use blood eosinophils as surrogate of sputum eosinophils to evaluate aireway inflammation could be misleading since some asthmatic patients present only local inflammation 10.

In conclusion, non-invasive methods such as induced sputum have been successfully introduced in clinical practice and research to study airway inflammation involved in the pathogenesis of respiratory diseases.

REFERENCES

1. Pizzichini E et al . Safety of sputum induction. Eur Respir J Suppl. 2002; 37:9s-18s 2. Paggiaro PL, et al. Sputum induction. Eur Respir J Suppl. 2002 Sep;37:3s-8s 3. Efthimiadis A, et al. Eur Respir J Suppl. 2002 Sep;37:19s-23s

95 4. Spanevello A, et al. Induced sputum to assess airway inflammation: a study of reproducibility. Clin Exp Allergy 1997; 27: 1138-1144 5. Spanevello A, et al. Induced sputum cellularity. Reference values and distribution in normal volunteers. Am J Respir Crit Care Med 2000; 62: 1172-1174. 6. Kips JC, et al. The use of induced sputum in clinical trials. Eur Respir J Suppl. 2002 Sep; 37:47s- 50s. 7. Balzano G, et al. Eosinophilic inflammation in stable chronic obstructive pulmonary disease. Relationship with neutrophils and airway function. Am J Respir Crit Care Med. 1999; 160:1486- 92 8. Bafadhel M, et al. Acute exacerbations of chronic obstructive pulmonary disease: identification of biologic clusters and their biomarkers. Am J Respir Crit Care Med. 2011; 184:662-71 9. Korevaar DA, et al. Diagnostic accuracy of minimally invasive markers for detection of airway eosinophilia in asthma: a systematic review and meta-analysis. Lancet Respir Med. 2015; 3:290-300. 10. Schleich FN, et al. Importance of concomitant local and systemic eosinophilia in uncontrolled asthma. Eur Respir J. 2014; 44:97-108.

EVALUATION

1. A pre-treatment with short acting 2 agonist a. Reduces the bronchoconstriction caused by the inhalation of hypertonic saline solution b. Increases the amount of sputum collection c. Avoids contamination of the collected sample d. It is useless

2. You can always perform the induction independently of patient’s FEV1 a. Yes b. No c. It depends on the smoking history of the patient d. None of the previous response

3. The amount of total sputum cells should be expressed as a. Cells/ml b. Cells/mg c. Both the previous response d. None of the previous response

4. Sputum sample of a healthy subject is mainly composed by: a. macrophages b. neutrophils c. eosinophils d. epithelial cells

5. Sputum analysis in COPD patients is mainly useful to: a. Predict reduction in FEV1 b. Evaluate severity of the disease c. Evaluate the effects of smoking d. Evaluate eosinophilic inflammation and predict response to corticosteroid therapy

96 EDUCATIONAL SKILLS WORKSHOP RESPIRATORY SAMPLING TECHNIQUES WORKSTATION 3: SPUTUM COLLECTION

Patrizia Pignatti 1 – Antonio Spanevello 2-3

1 Allergy and Immunology Unit, Fondazione Salvatore Maugeri, IRCCS Pavia, Italy 2 Division of Pneumology, , Salvatore Maugeri Foundation IRCCS, Tradate, Italy, 3 Department of Clinical and Experimental Medicine, University of Insubria, Varese, Italy

97 Conflict of interest disclosure

 We have no, real or perceived, direct or indirect conflicts of interest that relate to this presentation.

AIMS

• Aim 1: Present induction and processing method

• Aim 2: Discuss pros and cons of this methodology

• Aim 3: Show practical application cases

99 •Give the patient detailed information •Check safety of the equipment ultrasonic nebulizer (output ~ 1ml/min)

•Measure pre-bronchodilator FEV1 •Pre-treat with 200 mg of salbutamol

•After 10 min measure FEV1

•Start inhalation with a fixed concentration of hypertonic solution (3% or 4.5%) or use increasing concentrations (3, 4 5%).

•Measure FEV1 after each inhalation period. STOP if there is a FEV1 fall ≥ 20% of post-bronchodilator value

•Ask the patient to cough and spit after 5, 10, 15 and 20 min or whenever they have the urge to do so 100 ALTERNATIVE METHOD OF SPUTUM INDUCTION

•Start inhalation with 0.9% saline solution induction for 30 sec, 1 and 5 min measuring FEV1 after each period

•If no sputum production and no bronchoconstriction increase to 3-4.5% hypertonic solution

Additional recommendations

•Maintain the same protocol in a given study

•Do not repeat induction within 48h of the first induction

•Keep strictly to safety recommendations

101 ERS GUIDELINE RECOMMENDATIONS FOR SAFETY • Sputum induction should be performed by adequately trained technicians

• A physician should be present

• Hygiene and sterility should be assured

• Pre-treatment with short acting b 2 agonist

• Accepted both standard concentration of hypertonic saline (3 or 4,5%) 15 or 20 min. or increasing concentrations (from 3 to 4 or 5%). Modified protocol0.9% saline

• No data on safety of sputum induction in subjects with FEV1 < 1L or <50% pred.

• Maintain the same protocol of induction (hypertonic and duration) for the same subject for all the subsequent evaluations

• FEV1 checked after each inhalation period

102 Methods of sputum processing for cell counts, immunocytochemistry and in situ hybridisation. Efthimiadis A1, Spanevello A, Hamid Q, Kelly MM, Linden M, Louis R, Pizzichini MM, Pizzichini E, Ronchi C, Van Overvel F, Djukanović R.

Whole sputum sample Plug selection

103 Selection with the use of inverted microscope

104 ERS GUIDELINES - PROCESSING: KEY POINTS

1. Complete solubilization of the sample 2. Filter the sample to remove debris 3. Perform manual total cell count prior to centrifugation 4. Count the entire volume of the counting chamber 5. Prepare cytospin with adequate cell amount 6. Optimize buffer and stains 7. Perform a 400-nonsquamous cell differential count 8. Report squamous cells separately 9. Include negative and positive controls for special stains 10.Implement a regular quality control system 11.Use standard operating procedures

105 ERS GUIDELINES RECOMMENDATIONS FOR DIFFERENTIAL CELL COUNT

 Total cell count and viability  Differential cell count: count at least 500 non squamous cells

 Calculate the percentage of each cellular component (macrophages, neutrophils, eosinophils, lymphocytes and epithelial cells) with respect to the total cell count. Squamous cells must be counted separately.

Squamous cell  Calculate the percentage of squamous cells with respect to the total cell count Saliva contamination

106 PROCESSING SPUTUM SAMPLES

Smearing of sputum sample Cytospin of processed sputum sample

107 CELL STAINING AND ANALYSIS

►Diff-Quick

►May Grunwald-Geimsa

Metachromatic ►Toluidine blue cells http://pathology.mc.duke.edu/research

►Oil red

►PERLS

108 DIFFERENTIAL CELL COUNT NORMAL VALUES

 Macrophages 69.2 ± 13.0%

 Neutrophils 27.3 ± 13.0%

 Eosinophils 0.6 ± 0.8%

 Lymphocytes 1.0 ± 1.2%

 Epit. cells 1.5 ± 1.8%

Spanevello A et al. Am J Respir Crit Care Med 2000 109 INFORMATION COMING FROM TOTAL CELL COUNT AND VIABILITY

 High total cell count  Possible viral or bacterial infection

High viability -> Recent and rapid cell recruitment

Low viability  -“old” material -Bronchial epithelial cell-rich material -Patient treated with corticosteroids (for eosinophils)

110 Salive contamination of the sample When the sample is non-idoneous?

>20-30% squamous cells (plug selection method)

>70% squamous cells (in toto method) 111 EFFECT OF THE SMOKING HABIT ON SPUTUM CELLS (I)

Antracotic inclusion in macrophage cytoplasm

This type of deposits remains for long time in the cytoplasm of macrophages even after smoking quitting

112 negative ANTRACOTIC INCLUSIONS

PERLS positive Staining

113 EFFECT OF THE SMOKING HABIT ON SPUTUM CELLS (II)

Increase in sputum neutrophils

Sputum neutrophilia usually reduces 6 weeks after smoking quitting

Hargreave FE

114 ASTHMATIC SUBJECT

Eosinophils

Apoptotic eosinophils

115 CHARCOT LEYDEN CRYSTALS

116 Asthma Eosinophils - onset - follow up (corticosteroid therapy) - exposure to the sensitizing agent - exacerbations (also after viral infections)

Eosinophilic bronchitis

Neutrophils Airway infections Severe asthma – resistent to steroids COPD Pollution exposure Smoking, etc.. 117 SCARCE SPUTUM SAMPLES The International Guidelines for sputum processing, as well as other published studies, have left the question open as to whether there should be a weight threshold for the portions collected, below which the analysis loses significance and reproducibility.

Balbi B, Pignatti P, Corradi M, et al

Sputum samples with low viability

Low viability of the sample could affect total and differential cell count

Use of inhaled corticosteroids particularly decrease eosinophil viability

Apoptotic or damaged eosinophils are much more recognizable than apoptotic neutrophils 118 WHAT PERCENTAGE OF SPUTUM EOSINOPHILS SHOULD WE CONSIDER SIGNIFICANT FOR AIRWAY EOSINOPHILIA?

The cut-off value for sputum eosinophilia varies from 2-3% according to different authors and to ERS sputum Guidelines

Assessment and reproducibility of non-eosinophilic asthma using induced sputum Simpson JL, et al. Respiration 2010

All cut-points greater than 2% eos were reproducible

3% cut-point resulted in the highest agreement to distinguish eosinophilic from

non-eosinophilic airway inflammation in asthma 119 APPLICATION OF INDUCED SPUTUM (I)

Stability of airway inflammation phenotypes:

• Repeat evaluation of airway inflammation after 6mts/1year

• In stable condition reduction of airway inflammation • Exacerbation  increase airway inflammation

• Viral or bacterial infection partly responsible for the change in inflammatory pattern

120 APPLICATION OF INDUCED SPUTUM (II)

Bronchial inflammation can mirror nasal inflammation:

Consider nasal inflammation

United airway disease

NASAL INFLAMMATION vs BRONCHIAL INFLAMMATION

121 APPLICATION OF INDUCED SPUTUM (III)

Bronchial inflammation pre and post specific inhalation challenge:

When is it important to precisely determine sputum cells count?

PRE specific inhalation challenge (SIC) POST specific inhalation challenge (SIC)

Eosinophils

122 APPLICATION OF INDUCED SPUTUM (IV)

Identification of Eosinophilic Bronchitis

Asthma Eosinophilic bronchitis Respiratory Dyspnea, cough, wheezing, chest Cough symptoms thigtness Pulmonary function Normal or reduced Normal test (FEV1) Reversibility test Frequently positive (>12%) Negative Methacholine test Frequently positive (PD20<1000 mg) Negative Chest X ray Frequently normal Normal Atopy Present/Absent Present/Absent

Sputum eosinophils Frequently present Present as unique alteration

123 APPLICATION OF INDUCED SPUTUM (IV)

Eosinophilic airway inflammation in COPD patients

31 patients with stable mild-to-moderate COPD

Zanini A, et al. Int J COPD 2015 124 Conclusion

•Sputum collection and evaluation is an useful method to assess airway inflammation.

•It can be easly done by trained personal

125 Sputum collection Information of best practice

Dr Patrizia Pignatti Allergy and Immunology Unit Fondazione Salvatore Maugeri Via Salvatore Maugeri 10 27100 Pavia ITALY [email protected]

Keep strict to International Guidelines both for induction and processing

Induction:

1. Sputum induction should be performed by adequately trained technicians 2. A physician should be present 3. Hygiene and sterility should be assured 4. Pre- 5. Accepted both standard concentration of hypertonic saline (3 or 4,5%) 15 or 20 min. or increasing concentrations (from 3 to 4 or 5%). Modified protocol0.9% saline 6. No data on safety of sputum induction in subjects with FEV1 < 1L or <50% pred. 7. Maintain the same protocol of induction (hypertonic and duration) for the same subject for all the subsequent evaluations 8. FEV1 checked after each inhalation period

Processing:

1. Complete solubilization of the sample 2. Filter the sample to remove debris 3. Perform manual total cell count prior to centrifugation 4. Prepare cytospin with adequate cell amount 5. Optimize buffer and stains 6. Perform a 400-nonsquamous cell differential count 7. Report squamous cells separately 8. Include negative and positive controls for special stains 9. Implement a regular quality control system 10. Use standard operating procedures

126 Handling sputum induction with other functional and/or inflammatory evaluations

1. Methacholine challenge can be performed 1h before sputum induction without interference in inflammatory sputum cells (Spanevello A et al. Thorax 1999) 2. Sputum induction with hypertonic, but not isotonic, saline led to a marked decrease in FeNO not related to a fall in FEV1 following sputum induction (Beier J et al. ERJ 2003) 3. Hypertonic saline solution affects the evaluation of some cytokines in exhaled breath condensate (Carpagnano GE, et al. Chest 2005)

127 Analysis of bronchoalveolar lavage (BAL) fluid

Prof. Dr Ulrich Costabel Ruhrlandklinik Dept Pneumology/Allergy Tueschener Weg 40 45239 Essen GERMANY [email protected]

AIMS

 To describe the technique and laboratory processing of bronchoalveolar lavage (BAL) in the diagnostic work-up of diffuse lung disease  To explain how the results of BAL cytology should be interpreted in accordance with clinical information  To provide the basis for understanding the sensitivity and specificity of BAL findings for major diffuse lung disease: when is BAL alone sufficient, when is biopsy needed?

SUMMARY

Since its first application in the 1970s as a valuable research tool to study local immune and inflammatory mechanisms, bronchoalveolar lavage (BAL) has gained widespread acceptance as a powerful investigative tool in pulmonary medicine and has become a standard diagnostic procedure in patients with interstitial lung disease (ILD). BAL is used to collect cells, inhaled particles, infectious organisms and solutes from the lower respiratory tract and, in particular, from the alveolar spaces of the lung.

Diagnostic BAL serves as a "window to the lung". BAL is regarded as providing complimentary information to histopathology from biopsies, but nevertheless has several advantages over biopsy procedures. It is safe and associated with virtually no morbidity. Thus, it can be used repeatedly to investigate serial changes. In addition, BAL collects samples from a much larger area of the lungs than can be obtained by the small tissue fragments of transbronchial biopsy or even by surgical biopsy specimen, therefore giving a more representative view of inflammatory and immunological changes.

Technical aspects

Guidelines and recommendations for a standardised approach to the performance of BAL, and the laboratory processing and the analysis of recovered fluid have been published (1-6). When the guidelines are followed, the results of lavage are sufficient for practical diagnostic purposes.

Bronchoscopic procedure

BAL is usually performed during fibreoptic bronchoscopy (FOB) with topical anaesthesia, following general inspection of the tracheobronchial tree. The fibreoptic bronchoscope is gently introduced until impacted, or "wedged", in a segmental or subsegmental bronchus. In diffuse lung disease, the middle or lingular lobe is recommended as a standard site of BAL. Localised disease naturally requires lavage of the radiographically involved area.

128 Commonly, sterile, unbuffered isotonic saline (0.9 % NaCl solution) is used as the instillate. The fluid is instilled with syringes through the biopsy channel of the bronchoscope, using multiple aliquots of 20–60 mL (usually four to five aliquots), up to a total volume of 100–300 mL. Smaller instilled volumes (<100 mL) increase the likelihood of contamination of lavage fluid by the bronchial spaces, including inflammatory cells from this compartment, which may influence the differential cell counts. After each instillation, the aliquot is immediately recovered by gentle hand suction into the syringe or gentle wall suction into a fluid trap. Usually, 40–70 % of the instilled volume is recovered. The total volume retrieved should be greater than 30% of the instilled volume in order to avoid a misleading cell differential.

BAL is generally well tolerated. Side-effects are more or less those of routine FOB under local anaesthesia. There is practically no mortality, and the complication rate is low, ranging 0–2.3% compared to 7 % with transbronchial biopsy and 13 % with surgical lung biopsy (7- 10). There are no absolute contraindications for performance of BAL beyond those noted for bronchoscopy.

Laboratory processing

The total recovered fluid should be transferred to the laboratory as quickly as possible, since the cells are not well preserved in the saline solution. The total number of cells are counted in a haemocytometer, either in a sample of the pooled native fluid or in a re-suspension of the cells after the first centrifugation. Cell viability is assessed by trypan blue exclusion and should range 80–95%.

For the enumeration of cell differentials, 600 cells are counted after staining with May- Grünwald-Giemsa stain. A high percentage of epithelial cells (>5%) is indicative of contamination of the alveolar samples by bronchial cells. Such BAL probes may not be representative for the diagnosis of diffuse parenchymal lung disease. A number of soluble components have been measured in BAL fluid. None of them has proven to be useful in clinical settings (4, 5)

Normal findings in healthy adults without lung disease

The BAL fluid obtained from healthy, non-smoking adults without lung disease contains only small percentages of lymphocytes, neutrophils and other inflammatory cells. Alveolar macrophages are the predominant cell population.

Cigarette smoking is a strong confounding factor with significant effects on BAL samples. The alveolar macrophages from smokers are much larger than those in non-smokers, and contain cytoplasmic inclusion bodies (smoker´s inclusion bodies). On gross examination, the recovered BAL fluid has a light to dark brown and turbid appearance caused by the colour of the tar-laden macrophages. The total cell yield is three to five fold higher in smokers, due to a three to five fold numerical increase in the number of macrophages, leading to a relative decrease of the percentage of lymphocytes.

The normal values of differential cytology following BAL that have been proposed in the literature are somewhat variable. In most of the published studies there are only small numbers of normal patients/persons (mostly volunteers) available for comparison. For practical reasons, the following percentages can be expected as normal in nonsmokers (2):  macrophages >80%  lymphocytes 15%  neutrophils 3%  eosinophils 0.5%  mast cells 0.5 %

129 BAL as a clinical diagnostic tool

BAL is broadly indicated in every patient with unclear ILD or unclear pulmonary shadowing, no matter what cause is suspected. The underlying disorders may be of infectious, non- infectious, immunological or malignant aetiology. BAL may also be indicated in patients with normal chest radiograms when clinical and lung function tests are abnormal and point toward a diffuse lung disease, or in patients with unexplained pulmonary symptoms in whom a normal BAL finding may allow the exclusion of significant, active ILD.

Interpretation of BAL cytology

Changes in the profile of the cell differentials of BAL fluid have been described in various ILDs. Clearly, an abnormal BAL cell differential count does not allow a specific diagnosis, but BAL studies should not be limited to counting cell differentials. It is at least as important to note the morphological appearances of cells and particles. Furthermore, BAL cell differentials should not be used as an isolated finding for making a diagnosis, but should always be interpreted in the context of disease history, and clinical, laboratory and radiological findings.

BAL in differential diagnosis of ILD

BAL has improved diagnostic work-up in ILD, and some investigators use BAL more frequently than transbronchial lung biopsy. In two international statements on the major interstitial lung diseases, BAL was considered helpful in strengthening the diagnosis in patients with sarcoidosis in the absence of biopsy, and BAL and/or transbronchial biopsy were considered requirements for the exclusion of other diseases in a patient with IPF who did not undergo surgical biopsy (one of the four major criteria for making a clinical diagnosis of the disease) (11, 12). Recommendations for BAL were also included in the interstitial lung disease guideline of the British Thoracic Society (13).

BAL findings may be, on occasion, very specific, can directly confirm a particular diagnosis and then replace lung biopsy (table 1). It is of interest to note that many of these disorders are included in the group of the alveolar filling syndromes. Obviously, the abnormal material which accumulates in the alveolar spaces in these syndromes can easily be washed out by lavage and is so characteristic that a specific BAL diagnosis is possible.

In other selected lung diseases, BAL findings are not diagnostic, but may help narrow the differential diagnosis in the appropriate clinical setting (14). The list of diseases with a lymphocytic, neutrophilic, eosinophilic or a mixed cellular pattern is long (table 2). Diseases with an increase in lymphocytes can be further differentiated into those with an elevated, normal or decreased CD4/ CD8 ratio (table 3). However, the importance of the CD4/CD8 ratio has been debated because of the high variability in several diseases (14). Only 50-60% of sarcoidosis patients show an increased CD4/CD8 ratio, and in 15% the ratio is even decreased. In extrinsic allergic alveolitis, the ratio is not always decreased, as was seen in early BAL studies, but may also be normal or increased.

Some of the ILDs almost always show an abnormal BAL (high sensitivity), although the specificity is low; however, in combination with clinical and HR-CT features, the diagnosis may be possible without a biopsy (Table 4). For example, if the HR-CT scan shows a patchy ground glass pattern, BAL may be able to reveal that this patient suffers from extrinsic allergic alveolitis (high lymphocyte count), or a smoking related respiratory bronchiolitis/interstitial lung disease (high smoker macrophage count and normal cell differential), or alveolar hemorrhage (high count of hemosiderin laden macrophages). In drug- induced lung disease, BAL findings are not specific but can contribute to the expected

130 clinicopathologic pattern of a given drug-induced lung disease and can help to exclude other conditions such as infection or lung involvement by the underlying disease (15). Sometimes, even a normal lavage may be useful to exclude some disorders with high probability (e. g. extrinsic allergic alveolitis, eosinophilic pneumonia, alveolar hemorrhage) and to focus attention on other direction.

Assessment of disease activity and prognosis

It is not still established whether BAL is clinically useful for assessing the activity of disease processes and to provide prognostic information. Nevertheless, in IPF a recent retrospective analysis revealed that an increased baseline BAL neutrophil percentage is an independent predictor of early mortality whereas lymphocyte and eosinophil percentages are not (16). In idiopathic NSIP, the BAL lymphocyte count is higher and a BAL lymphocytosis is more frequently observed than in IPF, and this has been found to be associated with a longer survival (17).

It has not been proved that BAL is better than other parameters to guide therapy. In this regard, serial BAL to monitor the course of disease cannot be routinely recommended at present.

REFERENCES

1. Klech H, Hutter C, eds. Clinical guidelines and indications for bronchoalveolar lavage (BAL): report of the European Society of Pneumology Task Force on BAL. Eur Respir J 1990; 3: 937 – 974 2. Klech H, Pohl W, eds. Technical recommendations and guidelines for bronchoalveolar lavage (BAL). Report of the ERS Task Group. Eur Respir J 1989; 2: 561 - 585 3. BAL Cooperative Steering Group. Bronchoalveolar lavage constituents in healthy individuals, idiopathic pulmonary fibrosis, and selected comparison groups. Am Rev Respir Dis 1990; 141: S169 - S202 4. Haslam PL, Baughman RP, eds. Report of European Respiratory Society (ERS) Task Force: guidelines for measurement of acellular components and recommmendations for standardization of bronchoalveolar lavage (BAL). Eur Respir Rev 1999; 9: 25 – 157 5. Haslam PL, Baughman RP. Report of ERS Task Force: guidelines for measurement of acellular components and standardization of BAL. Eur Respir J 1999; 14: 245-248 6. Meyer KC et al. An official ATS clinical practice guideline: The clinical utility of BAL cellular analysis in interstitial lung disease. Am J Respir Crit Care Med 2012; 185: 1004- 1014 7. Costabel U, Guzman J. Bronchoalveolar lavage in interstitial lung disease. Curr Opin Pulm Med 2001; 7: 255 – 261 8. Reynolds HY. Use of bronchoalveolar lavage in humans – past necessity and future imperative. Lung 2000; 178: 271 – 293 9. Costabel U. CD4/CD8 ratios in bronchoalveolar lavage fluid: of value for diagnosing sarcoidosis? Eur Respir J 1997; 10: 2699-2700 10. Costabel U. Atlas of bronchoalveolar lavage. London, Chapman and Hall, 1998 11. Hunninghake GW, Costabel U, Ando M, et al. ATS/ERS/WASOG Statement on Sarcoidosis. Sarc Vasc Diffuse Lung Dis 1999; 16:149-173 12. ATS/ERS Statement. Idiopathic pulmonary fibrosis: Diagnosis and treatment. Am J Respir Crit Care Med 2000; 161: 646-664 13. Wells AU, Hirani N et al. Interstitial lung disease guideline: the British Thoracic Society in collaboration with the Thoracic Society of Australia and New Zealand and the Irish Thoracic Society. Thorax 2008; 63(Suppl V): v1-v58 14. Bonella F, Ohshimo S, Bauer P, Guzman J, Costabel U: Bronchoalveolar lavage. Eur Respir Mon 2010; 48: 59-72

131 15. Costabel U, Uzaslan E, Guzman J. Bronchoalveolar lavage in drug-induced lung disease. Clin Chest Med 2004; 25: 25-35 16. Kinder BW, Brown KK, Schwarz MI, et al. Baseline BAL neutrophilia predicts early mortality in idiopathic pulmonary fibrosis. Chest 2008; 133: 226-232 17. Ryu YJ, Chung MP, Han J, et al. Bronchoalveolar lavage in fibrotic idiopathic interstitial pneumonias. Respir Med 2007; 101: 665-660.

132 Table 1. Diagnostic BAL findings

BAL finding Diagnosis Pneumocystis carinii, fungi, CMV Opportunistic infections transformed cells

Milky effluent, PAS-positive Alveolar proteinosis noncellular corpuscles, amorphous debris, foamy macrophages

Haemosiderin-laden macrophages, Alveolar haemorrhage syndrome intracytosplasmic fragments of red blood cells in macrophages, free red blood cells

Malignant cells of solid tumours, Malignant infiltrates lymphoma, leukaemia

Dust particles in macrophages, Dust exposure quantifying asbestos bodies

Eosinophils >25% Eosinophilic lung disease

Positive lymphocyte transformation Chronic beryllium disease test to beryllium

CD1-positive Langerhans cells Langerhans cell histiocytosis increased

Atypical hyperplastic type II Diffuse alveolar damage, drug pneumocytes Toxicity

133 Table 2. BAL cellular patterns as an adjunct to diagnosis

Lymphocytic Extrinsic allergic alveolitis Berylliosis Sarcoidosis Tuberculosis NSIP (mainly cellular type) LIP Connective-tissue disorders Drug-induced pneumonitis Malignant infiltrates Silicosis Early asbestosis Crohn’s disease Primary biliary cirrhosis HIV infection Viral pneumonia Netrophilic (+/- eosinophilic) Idiopathic pulmonary fibrosis Desquamative interstitial pneumonia Fibrotic NSIP Acute interstitial pneumonia Acute respiratory distress syndrome Bacterial pneumonia Connective tissue disorders Asbestosis Wegener’s granulomatosis Diffuse panbronchiolitis Transplant bronchiolitis obliterans Idiopathic bronchiolitis obliterans Drug-induced reaction Eosinophilic Eosinophilic pneumonia Churg-Strauss syndrome Hypereosinophilic syndrome Allergic bronchopulmonary aspergillosis Idiopathic pulmonary fibrosis Drug-induced reaction Mixed cellularity BOOP Connective-tissue disorders NSIP Drug-induced reaction Inorganic dust disease

134 Table 3. CD4/CD8 ratio in diseases with lymphocytic alveolitis

CD4/CD8 increased CD4/CD8 normal CD4/CD8 decreased

Sarcoidosis Tuberculosis Extrinsic allergic alveolitis

Beryllium disease Lymphangitic Drug-induced pneumonitis carcinomatosis Asbestos-induced alveolitis BOOP

Alveolar proteinosis Silicosis

Crohn’s disease HIV infection

Connective-tissue disorders

135 Table 4. Diagnostic yield of BAL in ILD

BAL without biopsy usually sufficient (high sensitivity and high specificity) Alveolar proteinosis Pneumocystis pneumonia Bronchoalveolar carcinoma Alveolar hemorrhage Eosinophilic pneumonia

BAL in combination with clinical and HRCT features frequently sufficient (high sesitivity, low specificity) IPF (neutrophils  eosinophils) Extrinsic allergic alveolitis (lymphocytes, plasma cells, foamy macrophages) RB-ILD (smokers´macrophages) BOOP (mixed cellularity, CD4/CD8 ) Lymphangioleiomyomatosis (alveolar hemorrhage)

BAL typical in only 50% of patients, biopsy often needed (if CT atypical) (moderate sensitivity, high specificity) Sarcoidosis (CD4/CD8 ) Langerhans cell histiocytosis (CD1a>4%)

BAL mostly not diagnostic, biopsy required (low sensitivity  low specificity) Hodgkin´s disease Invasive aspergillosis

BAL = bronchoalveolar lavage; HRCT = high-resolution computed tomography; IPF= idiopathic pulmonary fibrosis; RB-ILD = respiratory bronchiolitis-associated interstitial lung disease; BOOP = bronchiolitis obliterans organizing pneumonia.

136 Analysis of bronchoalveolar lavage (BAL) fluid

Prof. Ulrich Costabel University of Duisburg-Essen Ruhrlandklinik Essen, Germany

137  I have no real or perceived conflicts of interest that relate to this presentation:

This event is accredited for CME credits by EBAP and EACCME and speakers are required to disclose their potential conflict of interest. The intent of this disclosure is not to prevent a speaker with a conflict of interest (any significant financial relationship a speaker has with manufacturers or providers of any commercial products or services relevant to the talk) from making a presentation, but rather to provide listeners with information on which they can make their own judgments. It remains for audience members to determine whether the speaker’s interests, or relationships may influence the presentation. The ERS does not view the existence of these interests or commitments as necessarily implying bias or decreasing the value of the speaker’s presentation. Drug or device advertisement is forbidden.

138 Bronchoalveolar Lavage

• Purpose: Differential diagnosis of interstitial lung disease or unclear parenchymal infiltrates

• Important: BAL is different from - bronchial washings used for bacteriology / tumor cytology (small volumes, 10 - 30 ml) - whole lung lavage used for alveolar proteinosis (large volumes, 10 - 40 liters)

139 Comparison of Biopsy/Cytology procedures

BAL TBB Open Biopsy Invasiveness + ++ +++ Days in 0 0-1 3-5 hospital Mortality 0% 0.1-0.2% 0.4-10%

140 BAL

 How to do it

141 Bronchoalveolar Lavage

Good local anesthesia

No coughing

No putride secretions

Wedge position

142 Technical recommendations and guidelines for bronchoalveolar lavage (BAL) • Report of the European Society of Pneumology Task Group on BAL Klech H, Pohl W (eds) Eur Respir J 1989; 2: 561-585 • Haslam P, Baughman R. Report of ERS Task Force. Eur Respir J 1999; 14: 245-248 • BAL Cooperative Steering Committee. Am Rev Respir Dis 1990; 141: S169-S202

143 Site of BAL

• diffuse disease: ML or lingula

• Patchy disease: involved segment

144 Methods to instil and recover the fluid

Instillation • Syringe, 20-50 ml bolus Recovery • Manual aspiration back into the syringe • Suction trap with mechanical suction

Total instillation volume: 100 - 300 ml Recovery rate > 30%

145 146 147 148 149 150 151 152 BAL Fluid Analysis

• Cell differentials (profile of inflammatory cells) • Immunocytology (CD4/CD8, CD1) • Cellular morphology: tumor cells, alveolar proteinosis, haemorrhage, foamy macrophages, plasma cells • Dust particles and fibres (e.g. asbestos bodies) • Infectious organisms

153 BAL

 How to read it

154 Normals:

Non-smokers and smokers

155 NORMAL NONSMOKER SUBJECT: YIELD APPROXIMATELY 10 MIO CELLS, WITH

> 85% Alveolar macrophages < 15% Lymphocytes < 3% Neutrophils < 0.5% Eosinophils < 0.5% Basophils, Mast cells.

156 Nonsmoker Smoker

157 BAL

 How to use it

158 BAL as a clinical tool

• Specific findings may replace biopsy • BAL cell differentials are nonspecific, but a useful adjunct to diagnosis in the appropriate clinical setting

• Value of BAL in assessment of prognosis and treatment efficacy: still an open question

159 Diseases that can be diagnosed by BAL alone (high sensitivity and specificity)

• Alveolar filling diseases (CT: ground glass, alveolar consolidation) - alveolar proteinosis - pneumocystis jiroveci - eosinophilic lung disease - alveolar haemorrhage - bronchoalveolar carcinoma

160 Alveolar Proteinosis

161 162 BAL may reveal infection: Pneumocystis

163 Toluidine Blue Stain

164 CMV Cell

165 BAL may reveal eosinophilic lung disease

166 167 BAL may reveal alveolar haemorrhage

168 BAL may reveal significant dust exposure

169 170 BAL may reveal asbestos exposure

171 172 173 174 BAL may reveal malignancy

175 Hyperplastic Type II Pneumocytes

176 Diagnostic yield of BAL in diffuse malignancy (Semenzato, Poletti, Respiration 1992)

• Bronchoalveolar carcinoma 93% • Adenocarcinoma 77% • Squamous cell carcinoma 50% • Non-Hodgkin lymphoma 67% • Hodgkin lymphoma 33% • Total (n=162) 76%

177 BAL as a clinical tool

• Specific findings may replace biopsy • BAL cell differentials are nonspecific, but a useful adjunct to diagnosis in the appropriate clinical setting

• Value of BAL in assessment of prognosis and treatment efficacy: still an open question

178 BAL pattern

• Neutrophilic IPF etc. • Lymphocytic Sarcoidosis, extrinsic allergic alveolitis, etc. • Eosinophilic CEP, Churg-Strauss-Syndrome, etc. • Mixed cellularity BOOP, etc.

179 Increase of neutrophils (+ eosinophils) in BAL • IPF • DIP • AIP • ARDS • Collagen vascular disease • Wegener´s granulomatosis • Asbestosis • Bronchopulmonary infections • etc...

180 Role of BAL in Diagnosis of IPF

• BAL may reveal alternative specific diagnoses: malignancy, infections, eosinophilic pneumonia, alveolar proteinosis • Increase in neutrophils +/- eosinophils (in 90%) suggests a fibrosing process: IPF, collagen/vascular disease, asbestosis • An increase in lymphocytes >30% is uncommon, exclude: EAA, BOOP, NSIP, LIP, sarcoidosis

181 Increase of lymphocytes in BAL

• Extrinsic allergic alveolitis • Sarcoidosis • Beryllium disease • Tuberculosis • BOOP • Drug induced diseases • Lymphangitic carcinomatosis • etc...

182 BAL Profile in Sarcoidosis

• Lymphocytes  in 90% of patients • Clinically active disease: Lymphocytes range 20~80%, mean ~40% • Clinically inactive disease: Lymphocytes lower, mean ~30%, but broad overlap • Neutrophils may be increased in late or advanced disease

183 BAL in Sarcoidosis

184 BAL in EAA

185 Lymphocytes (% of total cells)

186 Diagnostic value of BAL CD4/CD8 ratio for sarcoidosis

CD4/CD8 Sensitivity Specificity Author > 3.5 59% 92% Costabel Milan 1987 > 4.0 59% 96% Winterbauer Chest 1993 > 4.0 55% 94% Thomeer WASOG1997

187 Value of various cut-offs of CD4/CD8 ratios for the diagnosis of sarcoidosis

CD4/CD8 ratio Sensitivity (%) Specificity (%)

2.0 80 73

3.5 54 93

5.0 38 98

10.0 12 100

Sarc. n=129; Non-sarc. n=353 Costabel et al 1987

188 Value of various cut-offs of CD4/CD8 ratios for the diagnosis of sarcoidosis

CD4/CD8 ratio Sensitivity (%) Specificity (%)

2.0 80 73

3.5 54 93

5.0 38 98

10.0 12 100

Sarc. n=129; Non-sarc. n=353 Costabel et al 1987

189 BAL advantages in sarcoidosis

• Low risk

• narrows differential diagnosis

• supports the diagnosis (lymphocytes , CD4/CD8 ) in up to 70% of patients

• Characteristic BAL plus characteristic clinical presentation may obviate biopsy

190 BAL profile in EAA

• Very high total cell count (> 4 fold) • Very high lymphocyte % (> 50%) • Mild increase in neutrophil % and mast cells • Presence of plasma cells • Presence of foamy macrophages • CD4/CD8 usually < 1.0 (but may be normal or increased) • Profiles may change with chronic disease

191 EAA and BAL CD4/CD8 ratio

Barrera L et al. AJRCCM 2008 177: 44-55 192 Histological pattern in chronic pigeon breeder’s disease: correlation with clinical data

Typical pattern NSIP pattern UIP-like pattern n = 58 n = 22 n = 10 p

Finger clubbing 30/56 (53) 10/21 (47.6) 8/10 (80) 0.26

BAL lymphocytes % 65 ± 21 52 ± 23 36 ± 23 0.0011

BAL macrophages % 34 ± 20 45 ± 23 59 ± 18 0.0028

BAL eosinophils % 1 (0–9) 0 (0–13) 2 (0–13) 0.11

BAL neutrophils % 0 (0–10) 1 (0–10) 1 (0–4) 0.61

HRCT Inflammation (%) 30/40 (75) 11/16 (69) 1/7 (14) <0.007 Fibrosis (%) 10/40 (25) 5/16 (31) 6/7 (86) <0.007

Gaxiola M et al, Respir Med 2011 193 Clinical significance of BAL in EAA

• A normal BAL widely excludes EAA • BAL is the most sensitive tool in detecting signs of alveolitis • BAL cannot differentiate between symptomatic ILD and subclinical alveolitis in a given patient • In the follow-up, persistent BAL abnormalities indicate that complete allergen avoidance has not been achieved

194 Diseases which can largely be excluded by a normal BAL

• Extrinsic allergic alveolitis • Alveolar haemorrhage • Alveolar proteinosis • Any siginificant „active“ ILD

195 How to interpret BAL findings

• Not in isolation but in context with clinical presentation and HRCT findings!

196 197 Ground Glass Pattern on HR-CT

BAL findings Diagnosis • Lymphocytes > 50% Extrinsic allergic alveolitis Total cell count high • Eosinophils > 25% Eosinophilic pneumonia • Pneumocystis Infection • Iron laden macrophages Alveolar haemorrhage • Smoker´s macrophages DIP/RBILD + mild increase in neutros/eos

198 IPF

199 Reticular/linear Pattern on HR-CT

BAL findings Diagnosis • Neutros + eos, IPF (UIP) + lymphocytes < 30% Collagen disease Asbestosis • Lymphocytosis Chronic EAA + neutros, eos, mast cells

• Malignant cells Lymphangitic + lymphocytosis carcinomatosis

200 201 Nodular or reticulonodular pattern on HR-CT BAL findings Diagnosis • lymphocytosis, normal Sarcoidosis appearance of macrophages, CD4/CD8 n or • lymphocytosis, dust particles Silicosis in macrophages, CD4/CD8 n or • malignant cells, Lymphangitic ± lymphocytosis carcinomatosis

202 Langerhans cell histiocytosis (LCH)

203 Cystic Pattern on HR-CT

BAL findings Diagnosis • Smoker´s macrophages, LCH mild increase in neutros/eos, CD1a+ cells > 4% • Moderate to severe Lymphangioleio- alveolar haemorrage myomatosis

204 How to integrate BAL into the management of ILD patients

• Specific findings may replace biopsy • Cell differentials are nonspecific but a useful adjunct to diagnosis in the appropriate clinical setting • Value of BAL in assessment of prognosis and treatment efficacy: still an open question

205 Role of BAL in ILD

• In idiopathic interstitial pneumonia : BAL differential may guide choice of treatment and determine prognosis?

206 BAL cells in NSIP vs IPF

Author Lymphocytes Neutrophils NSIP IPF NSIP IPF Nagai 37% 7% 8% 6% 1998 Daniil 9% 8% 8% 10% 1999 Park 46% 8% 23% 13% 2000 Suga 21% 6% 7% 7% 2000

207 Baseline BAL Neutrophilia Predicts Early Mortality in Idiopathic Pulmonary Fibrosis (%)

N= 156 survival Overall

Follow-up Time (years) Kinder BW et al. Chest 2008 208 Lymphocytosis on BAL in Fibrotic NSIP and IPF and Prognosis ALL IIP (N=122) NSIP (N=35) and IPF (N=87)

Ryu YJ, et al. Respir. Med. 2007 209 What the guidelines tell us

• ATS/ERS IIP Statement 2002

• New IPF Guidelines 2011

• Sarcoidosis Statement 1999

• BTS ILD Guideline 2008

210 ATS/ERS Statement 2002:

HRCT

Confident CT Atypical clinical Features diagnostic Suspected diagnosis of IPF with or CT features of another other DPLD consistent clinical for IPF DPLD e.g. HX features TBBx or If non-diagnostic TBBx, BAL or BAL? other relevant test

Surgical lung biopsy

UIP NSIP RB DIP DAD OP LIP non-IIP

confirmed 211 Diagnostic algorithm for IPF Am J Respir Crit Care Med 2011

Suspected IPF

Yes Identifiable causes

Nofor ILD?

HRCT PossiblePossible UIP UIP * UIP * InconsistentInconsistent w/ with UIP UIP *

Surgical Lung Not UIP † UIPBiopsy † ProbableUIP / Probable UIP † // Possible UIPUIP † NonNon--classifiableclassifiable fibrosisfibrosis †

MDD

IPF IPF/Not IPF per Table 4 Not IPF 212 Diagnostic algorithm for IPF Am J Respir Crit Care Med 2011

Suspected IPF

Yes Identifiable causes

Nofor ILD?

HRCT PossiblePossible UIP UIP * UIP * WhereInconsistentInconsistent w/ withis UIP UIP * the

BAL/TBBSurgical Lung ?? Not UIP † UIPBiopsy † ProbableUIP / Probable UIP † // Possible UIPUIP † NonNon--classifiableclassifiable fibrosisfibrosis †

MDD

IPF IPF/Not IPF per Table 4 Not IPF 213 Diagnosis of IPF

Should BAL cellular analysis be performed in the diagnostic evaluation of suspected IPF? Recommendation: weak against (low) - should not be done in the majority, but may be appropriate in a minority (vote: 4 for use, 18 against use, 1 abstention) Should transbronchial lung biopsy be used in the evaluation of suspected IPF? Recommendation: weak against (low) (vote: none for use, 23 against use, no abstentions)

Am J Respir Crit Care Med, 2011 214 Should BAL be performed in IPF ?

• BAL cellular analysis should be considered in the evaluation of patients with IPF at the discretion of the treating physician based on availability and experience at their institution/regional laboratory. • In the evaluation of patients with suspected IPF, the most important application of BAL is in the exclusion of chronic hypersensitivity pneumonitis; prominent lymphocytosis (>40%) should suggest the diagnosis.

Am J Respir Crit Care Med, 2011215 Significance of Bronchoalveolar Lavage for the Diagnosis of Idiopathic Pulmonary Fibrosis

Shinichiro Ohshimo, Francesco Bonella, Ai Cui, Martin Beume, Nobuoki Kohno, Josune Guzman, and Ulrich Costabel

Am J Respir Crit Care Med 2009; 179: 1043-1047

216 Suspicious IPF based Study Design on confident HRCT findings Application of and Results n = 101 ATS/ERS criteria 2002 Eligible Patients n = 74 Addition of BAL cell differential No analysis Lymphocytosis in BALF (cut off 30%) Yes No

n = 68 n = 6

Granulocytosis Granulocytosis in BALF in BALF (cut off 3%) (cut off 3%) Yes No Yes No

n = 63 n = 5 n = 4 n = 2 IPF IPF NSIP 3; 1 EAA EAA 217 Wells AU, Thorax 2008

218 Recommendations for BAL in ILD

• BAL or TBLB, when required, should be performed before the initiation of treatment. [D]

• BAL should be considered in all patients with suspected infection, malignancy and some rare ILDs. In such cases, BAL may be diagnostic. [C]

• BAL is not required as a diagnostic tool in patients with clinical features and HRCT appearances typical of IPF. [C]

219 Recommendations for BAL in ILD

• In patients for whom the diagnosis is uncertain after clinical assessment and HRCT scanning, typical BAL cellular profiles may allow a diagnosis of HP or sarcoidosis to be made with greater confidence. [C]

• In cases in which the diagnosis is uncertain and BAL is considered, the procedure should be performed in a regional centre with technical expertise in the procedure and the analysis of the BAL samples. [D]

• BAL should be performed in all patients undergoing TBLB. [D]

220 Am J Respir CritMeyer Care KC Med et al., 2009; BAL 179:Cinical 1043 Practice-1047 Guideline,

AJRCCM 2012 221 Summary: BAL contribution to ILD

• Specific diagnosis is possible.

• Level of confidence in a certain diagnosis can be enhanced or weakened.

• BAL and HR-CT combined may obviate the need for biopsy.

• BAL is included as a diagnostic tool in major guidelines on ILD.

222 Thank you for your attention Interstitial and Rare Lung Disease Unit

Prof. U. Costabel Dr. F. Bonella Dr. T.E. Wessendorf Dr. E. Börner Dr. X. Long Prof. J. Guzman Dr. S. Ohshimo Prof. D. Theegarten

223 Recommended reading list and E-learning resources

WORKSTATION 1 - Clinical cytology

WORKSTATION 2 - Histology

WORKSTATION 3 - Sputum collection

1. Brightling CE. Clinical applications of induced sputum. Chest 2006; 129; 1344–1348. 2. European Respiratory Society Task Force Standardised methodology of sputum induction and processing. Eur Respir J 2002; 20: Suppl. 37, 1s-55s. 3. Pizzichini E, et al. Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements. Am J Respir Crit Care Med 1996; 154: 308-317 4. Spanevello A, et al. Induced sputum cellularity. Reference values and distribution in normal volunteers. Am J Respir Crit Care Med 2000; 62: 1172-1174. 5. Spanevello A, et al. Induced sputum to assess airway inflammation: a study of reproducibility. Clin Exp Allergy 1997; 27: 1138-1144. 6. Spanevello A, Beghé B, Bianchi A, et al. Comparison of two methods of processing induced sputum: selected versus entire sputum. Am J Respir Crit Care Med. 1998; 157:665-8 7. Pizzichini MM, Popov TA, Efthimiadis A, et al. Spontaneous and induced sputum to measure indices of airway inflammation in asthma. Am J Respir Crit Care Med. 1996; 154:866-9 8. Bhakta NR, Woodruff PG. Human asthma phenotypes: from the clinic, to cytokines, and back again. Immunol Rev. 2011; 242:220-32. 9. Louis R, Lau LC, Bron AO, et al. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med. 2000; 161:9-16 10. Romagnoli M, Vachier I, Tarodo de la Fuente Pet al. Eosinophilic inflammation in sputum of poorly controlled asthmatics. Eur Respir J. 2002;20:1370-7 11. Louis R, Sele J, Henket M, et al. Sputum eosinophil count in a large population of patients with mild to moderate steroid-naive asthma: distribution and relationship with methacholine bronchial hyperresponsiveness. Allergy. 2002;57:907-12. 12. Pignatti P, Zanini A, Della Patrona S, Gumiero F, Cherubino F, Spanevello A. Sputum and exhaled breath analysis. ERS Handbook: Respiratory Medicine ERH-0025-2012.3d 2014 13. Green RH, Brightling CE, McKenna S, et al. Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet. 2002; 360:1715-21. 14. Grootendorst DC, Gauw SA, Verhoosel RM, et al. Reduction in sputum neutrophil and eosinophil numbers by the PDE4 inhibitor roflumilast in patients with COPD. Thorax. 2007; 62:1081-7. 15. Bafadhel M, McKenna S, Terry S, et al. Acute exacerbations of chronic obstructive pulmonary disease: identification of biologic clusters and their biomarkers. Am J Respir Crit Care Med. 2011;184:662-71 16. Schleich FN, Chevremont A, Paulus V, Henket M, Manise M, Seidel L, Louis R. Importance of concomitant local and systemic eosinophilia in uncontrolled asthma. Eur Respir J. 2014; 44:97- 108. 17. Korevaar DA, Westerhof GA, Wang J, Cohen JF, Spijker R, Sterk PJ, Bel EH, Bossuyt PM. Diagnostic accuracy of minimally invasive markers for detection of airway eosinophilia in asthma: a systematic review and meta-analysis.Lancet Respir Med. 2015; 3:290-300.

WORKSTATION 4 - Analysis of BAL fluids

1. U. Costabel, Atlas of bronchoalveolar lavage, Thieme publisher, 1994

224 Faculty disclosures

Prof. Helmut H. Popper has received unrestricted research grants from Eli Lilly (IALT-BIO), Astra Zeneca, Pfizer, Hofman La Roche, Boehringer-Ingelheim, as well as advisory board honoraria from Hofman La Roche, Eli Lilly, Boehringer-Ingelheim, Novartis and Pfizer.

Dr Erik Thunnissen has received honoraria or consultation fees from Eli - Lilly that supported meeting European experts 2010.

225 Faculty contact information

Prof. Dr Ulrich Costabel Prof. Antonio Spanevello Ruhrlandklinik Division of Pneumology Dept Pneumology/Allergy Salvatore Maugeri Foundation IRCCS Tueschener Weg 40 Via Roncaccio 16 45239 Essen 21049 Tradate GERMANY ITALY [email protected] Department of Clinical and Experimental Medicine Prof. Christian Kaehler University of Insubria Department of Internal Medicine Via Guicciardini 9 Innsbruck Medical University 21100 Varese Anichstrasse 35 ITALY 6020 Innsbruck [email protected] AUSTRIA [email protected] Dr Eva Schmutz Thoraxklinik at University of Heidelberg Dr Patrizia Pignatti Amalienstrasse 5 Allergy and Immunology Unit 69126 Heidelberg Fondazione Salvatore Maugeri GERMANY Via Salvatore Maugeri 10 [email protected] 27100 Pavia ITALY Dr Erik Thunnissen [email protected] Pathologie VU University Medical Center Prof. Helmut H. Popper De Boelelaan 1117 Pathology 1081 HV Amsterdam Medical University Graz NETHERLANDS Auenbruggerplatz 25 [email protected] 8036 Graz AUSTRIA Dr Matthias Wiebel [email protected] Thoraxklinik at University of Heidelberg Amalienstrasse 5 Prof. Dr Philipp A. Schnabel 69126 Heidelberg Instit. Alllgemeine&Spezielle Pathologie GERMANY University of Saarlandes [email protected] 66421 Homburg/Saar GERMANY [email protected]

226 Answers to evaluation questions

Please find all correct answers in bold below

WS2. Histology – Prof. Helmut H. Popper, Dr Erik Thunnissen

2. If a transbronchial biopsy did not yield in diagnostic tissue in lung cancer or an interstitial disease, what would you do next? a. repeat TBA b. proceed to VATS. c. perform EBUS TBNA d. perform BAL

3. Can an UIP diagnosis we made on cryobiopsies? a. no b. yes c. yes, but a differential diagnosis of underlying diseases is most often not possible d. no, the material is not sufficient

WS3. Sputum collection - Dr Patrizia Pignatti, Prof. Antonio Spanevello

2. You can always perform the induction independently of patient’s FEV1 a. Yes b. No c. It depends on the smoking history of the patient d. None of the previous response

3. The amount of total sputum cells should be expressed as a. Cells/ml b. Cells/mg c. Both the previous response d. None of the previous response

4. Sputum sample of a healthy subject is mainly composed by: a. macrophages b. neutrophils c. eosinophils d. epithelial cells