RESULTADOS SFT DE PACIENTES CON AR .1 Características de la población en estudio

RESULTADOS

IV.1 RESULTADOS SFT DE PACIENTES CON AR .1 Características de la población en estudio

Patricia L. Haslam

What it is and when to use it

Bronchoalveolar lavage (BAL) involves using a fibreoptic bronchoscope to wash a subsegment of the lungs with sterile physiological saline to sample components from the peripheral air spaces in health and disease. These include immune and inflammatory cells, other pathological cells or features, cytokines, enzymes, lipids or other secreted products, inhaled

environmental or occupational agents, and infections. Since the 1960s, BAL has been used extensively in research and to assist in the diagnosis of peripheral lung diseases, notably diffuse interstitial lung diseases (ILDs), occupational lung diseases, rare lung diseases, thoracic malignancies and lower respiratory tract infections (table 1).

Numerous publications, including guidelines from the European Respiratory

Society (ERS) and American Thoracic Society (ATS), confirm that BAL cytological or microbiological findings can often increase diagnostic confidence. However, BAL itself is rarely specifically diagnostic and must be interpreted together with clinical, physiological, radiological and other multidisciplinary investigations.

Prior to 2000, BAL was routinely included in the diagnostic work-up of parenchymal lung diseases. Currently, for ILDs, specialists consider that HRCT patterns are often sufficiently diagnostic to avoid the need for BAL or lung biopsy. An ATS/ERS consensus terminology for the idiopathic interstitial pneumonias published in 2002 has also changed the way specialists diagnose and manage this subgroup of ILDs. However, BAL is still indicated whenever the preliminary clinical investigations plus HRCT fail to establish a confident diagnosis, or where additional information is needed to confirm, strengthen or exclude a diagnosis.

How to obtain a sample

This section will only describe the

standardised BAL procedure recommended in Europe and BAL cytology methodology for investigation of adults with diffuse lung diseases where infection is not suspected.

A modified BAL procedure is used for the specialist diagnosis of lower respiratory tract infections, designed to minimise contamination with irrelevant microorganisms and to target sites of maximal involvement.

For both research and routine applications, a standardised BAL procedure must be followed in order to minimise variability due to the unknown dilution factor during lavage Key points

N

BAL is used to sample immune and inflammatory cells and many other components from the peripheral air spaces of the lungs in health and disease.

N

BAL is mainly used in research and to assist in the clinical diagnosis of ILDs or lower respiratory tract infections.

N

BAL findings must be interpreted in conjunction with results from clinical, pathological and radiological investigations.

N

A standardised procedure must be followed.

Table1.AguidetomaintypesofBALinflammatorycellsandothercytologicalfeaturesinlowerrespiratorydiseases PredominantBALinflammatorycelltypes increasedcomparedwithnormalrange#Othercharacteristiccytologicalfeatures Lowerrespiratorytractinfections Community-acquired;nosocomialpneumoniaNeutrophilsveryhighinbacterialpneumoniasIntracellularbacteriainactivepneumonia. Identifybyspecialstains,culturesetc. OpportunisticinfectionsinAIDS;organtransplant recipientsandpatientsonchemotherapyNeutrophilsoftenmoderatelyincreasedPneumocystiscarinii Cytomegalovirus Fungalinfections Othersbyspecialstains,culturesetc. Thoracicmalignancies AdenocarcinomaorbronchoalveolarcellcarcinomaNotofdiagnosticvalueTumourcellsreadilydetectable Metastaticorlymphangiticspreadfrom nonpulmonarytumoursNotofdiagnosticvalueTumourcellsreadilydetectable B-celllymphomasLymphocytesoftenstrikinglyincreasedAbnormallymphocytesconsistentwithlymphoma canbedemonstratedbyspecialstains Hodgkin’slymphomaReed–Sternbergcells Rarelungdiseases AlveolarlipoproteinosisNotofdiagnosticvalueGlobulesoflipoproteinplusacellulardebris OriginalBALfluid‘milky’ PulmonaryhaemosiderosisMainlymacrophagescontainingparticles similartothoseinsmokingbutorange-brownMajorityofmacrophagesheavilyladenwith haemosiderin,Perlstainpositive PulmonaryLangerhanscellhistiocytosisMainlymacrophagescontaining smoking-relatedparticles.5%ofthecellsshowntobeLangerhanscells byCD1astainingorelectronmicroscopy Fibrosingmineraldustdiseases AsbestosisModerateincreasesinneutrophilswithorwithout increasedeosinophilsorlymphocytesAsbestosbodiesindicatingexposure TalcpneumoconiosisInsufficientinformationTalcbodiesindicatingexposure

Table1.Continued Hardmetallungdisease/giantcellinterstitial pneumoniaMildincreasesinneutrophilswithorwithout increasedeosinophilsorlymphocytesRefractileparticlesofhardmetalin macrophagesplusgiantcellsifalsogiantcell interstitialpneumonia Drug-inducedlungdiseases Amiodarone-inducedpneumonitisLymphocytesincreasedLargephospholipidinclusionsinmacrophages Numerouserythrocytesand‘bloody’fluidAcutealveolarhaemorrhageNotofdiagnosticvalue Drug-inducedeosinophilicpneumoniaEosinophilsveryhigh Otherpulmonaryeosinophilias IdiopathiceosinophilicpneumoniaEosinophilsveryhigh Allergicdiseases:asthma;Churg–Strauss syndrome;bronchopulmonaryaspergillosisMildtomoderateincreasesineosinophilsplus lymphocytes Parasiticinfections:schistosomiasis;StrongyloidesEosinophilsoftenhigh AcuterespiratorydistresssyndromeNeutrophilsveryhigh Idiopathicinterstitialpneumonias IdiopathicpulmonaryfibrosisModerateincreasesinneutrophilswithorwithout increasedeosinophils NonspecificinterstitialpneumonitisMildincreasesinlymphocytesplusneutrophils withorwithoutincreasedeosinophils CryptogenicorganisingpneumoniaModerateincreasesinlymphocytesplusneutrophils LymphoidinterstitialpneumoniaIncreasesinlymphocytes Respiratorybronchiolitis-associatedILDMainlymacrophagescontainingsmoking particlesplusafewneutrophils DesquamativeinterstitialpneumoniaMacrophagescontainingsmokingparticlesplus moderateincreasesinneutrophilswithorwithout increasedeosinophilsorlymphocytes AcuteinterstitialpneumoniaNeutrophilsveryhigh

and many other potential sources of variability. There is still no globally agreed standard for the general conduct of BAL in adults for cytological and other purposes, but the ERS has long promoted BAL standardisation in a series of European guidelines. In 1999, the ERS published consensus guidelines recommending a standardised BAL procedure to use in adults based on the most comprehensive review of sources of variability for measurement of BAL components yet undertaken. The aim of using optimal BAL standardisation to minimise variability is to improve the reliability of quantitative measurements of all components.

Minimising variability is an essential scientific requirement for research.

However, a 2012 ATS clinical practice guideline on the clinical utility of BAL in ILD, recommends that ‘the BAL target site be chosen on the basis of an HRCT performed before the procedure, rather than choosing a traditional BAL site.’ This did not achieve full consensus because of the disadvantage that it would reduce BAL standardisation before it is known whether moving away from a standard BAL site would change diagnostic interpretation. To avoid compromising BAL standardisation, more research is needed into ILDs to compare lavages from both the standard BAL site and the site selected on the basis of HRCT, to conclude whether there is any clinical advantage to be gained. For now, the established standardised BAL procedure should also continue to be employed in patients with diffuse bilateral lung diseases. The standard site is also required to study healthy controls or patients with apparently ‘normal’ lungs, and to reduce variability for research. The site recommendation differs for patients with localised lung diseases such as some malignancies or infections, where BAL is targeted to the site of maximal involvement.

A protocol using the European recommended standard BAL procedure is as follows.

1) Perform BAL under local anaesthesia using fibreoptic bronchoscopy as part of pre-treatment assessment.

Table1.Continued Systemicconnectivetissuediseases SystemicsclerosisModerateincreasesinneutrophilswithorwithout increasedeosinophilsorlymphocytes Sjo¨gren’ssyndromeModerateincreasesinneutrophilswithorwithout increasedlymphocytes Granulomatouslungdiseases" SarcoidosisModerateincreasesinlymphocyteswithor withoutmildincreasesinneutrophilsCD4/CD8ratiosincreasedinabouthalf ofpatients Hypersensitivitypneumonitis/extrinsicallergicalveolitisLymphocytesveryhigh Neutrophilsandmastcellsalsoincreasedafter recentexposure

CD4/CD8ratiosfrequentlydecreased ChronicberylliumdiseaseModeratetohighincreasesinlymphocytesCD4/CD8ratiosincreasedinnearlyall Lymphocytesproliferatetoberylliumsalts #:usingdifferentialpercentageBALcellcounts(seemaintextforfullexplanation);":excludinginfections.

2) Proceed initially as for routine fibreoptic bronchoscopy:

N

generally semisupine patient positioning;

N

pre-medication with a sedating compound;

N

local anaesthesia with lidocaine removing any excess prior to lavage.

3) For lavage, gently wedge the tip of the bronchoscope into an appropriate

subsegmental bronchus. The recommended standard site is right middle lobe in diffuse lung diseases and healthy controls, but the area of greatest radiographic abnormality in localised lung diseases.

4) Sequentially introduce then aspirate standard aliquots (4660 mL) of sterile physiological saline pre-warmed to body temperature through the application tube of the bronchoscope. Do not exceed total introduction volume of 240 mL.

5) Aspirate each aliquot, keeping dwell time to the minimum, using very low suction pressure (3.33–13.3 kPa/25–100 mmHg) to avoid airway collapse.

6) Collect the recovered fluid into a container to which cells are poorly adherent (e.g. siliconised glass or a non-cell adherent plastic designed for suspension tissue cultures).

7) Record the lavage site, total BAL fluid introduction volume and number of aliquots, and the total recovery volume.

8) Immediately send the BAL sample to the laboratory to enable processing to

commence within 1 h because BAL cells deteriorate rapidly in saline.

9) Also send a patient protocol with age, sex, provisional diagnosis and other factors that influence BAL findings including smoking history (current, ex- or nonsmoker), current medications and associated diseases.

10) If biopsies are needed, perform these after BAL to avoid contamination of BAL with blood or bronchial tissue debris.

BAL is safe and side-effects are low, the same as for fibreoptic bronchoscopy alone,

except for an increased risk of minor post-lavage pyrexia, which can be minimised by keeping total BAL introduction volumes to ,300 mL.

Processing of samples for cytology BAL cells deteriorate rapidly in saline and laboratory processing should commence a maximum of 1 h after BAL sample collection.

To delay deterioration, BAL cells should be transferred into serum-free minimum essential medium containing 25 mM HEPES buffer (MEM-HEPES), which maintains pH 7.2–7.4 in an open system.

Non-cell adherent containers and pipettes must be used for all laboratory procedures.

The processing procedure is as follows.

1) Measure the total volume of the BAL sample.

2) Record any abnormality in the gross appearance of the fluid,e.g. a milky appearance suggestive of alveolar

lipoproteinosis or a very bloody appearance suggestive of acute haemorrhagic

conditions.

3) Mix sample to ensure even suspension then divide into measured aliquots for different departments if required (e.g.

o20 mL for BAL cytology and flow cytometry, 10 mL for microbiology and 20 mL for electron microscopy).

4) For BAL cytology, the fluid aliquot should be mixed and a cell viability test conducted (e.g. trypan blue). Then, make a total count of nucleated cells (per mL) using an improved Neubauer counting chamber and white cell counting stain (e.g. Kimura stain).

If the original BAL sample is too dilute for an accurate cell count, the count should be performed after separating the cells by centrifugation and resuspending them at a higher concentration.

5) Centrifuge the BAL sample at low speed (3006g at 4^oC for 10 min) to separate the cells and other insoluble components from the supernatant fluid. Aspirate the supernatant and aliquot it for storage at -70^oC. Then, wash the BAL cell pellet in MEM-HEPES and resuspend it in a small

volume (1–2 mL) to achieve a more concentrated suspension. Perform a total cell count, and calculate the number of cells per mL and total in the original BAL fluid.

6) Adjust the volume of the cell suspension to a standard 1.5610⁶cells?mL^-1to make cytocentrifuge slide preparations. Use 100-mL aliquots (1.5610⁵cells) per slide (spin at 906g for 4 min). Prepare at least six slides per patient. After air drying, fix two slides in methanol (not formalin, which impairs staining of mast cells). Stain with May–

Gru¨nwald–Giemsa for differential cell counting. Use other slides for special stains(e.g. Gomori–Grocott silver stain for fungi andPneumocystis carinii, and Perl stain for haemosiderin-laden macrophages).

Mucus contamination of BAL samples, if very excessive, can cause serious technical problems in processing. When there is such heavy contamination from the upper airways, BAL results must be interpreted with caution. Mucus can be removed by filtering the lavage through cotton gauze or nylon mesh but this can cause loss of adherent cells, dust fibres and other components. An alternative to avoid such loss is to remove mucus by treating the BAL cell pellet with the mucolytic dithiothreitol.

Some workers consider that when BAL cells are in tissue culture medium, processing can be delayed for 24 h to enable long-distance transport to centralised processing centres. However, this is not advisable because granulocytes are short lived and apoptotic changes start within 9 h.

Therefore, it is advisable to transfer BAL cells into tissue culture medium within 1 h and make cytocentrifuge preparations within 1–4 h. Staining of air-dried preparations can be delayed foro24 h if necessary. It is essential that BAL is conducted by clinical and laboratory personnel who are highly trained in the procedure, applications and interpretation.

Differential cell counting and other cytological appearances

The standard approach to counting BAL cells in cytocentrifuge preparations is to express the count of each type as a

percentage of the total BAL cells (differential percentage cell count). This proportionate approach is not affected by the unknown BAL dilution factor.

Differential cell counts are performed and other cytological features identified by examining May–Gru¨nwald–Giemsa-stained cytocentrifuge slide preparations by light microscopy. First, low-power magnification (610 and 625 objective lenses) is used to search the entire preparation and semi-quantitatively grade (on a scale from 0 to 5) any mucus and erythrocytes, and identify any unusual cytological features, such as inorganic dust particles or fibres, globules of lipoprotein, giant cells, malignant cells or microorganisms. Secondly, higher-power magnification (640 or 660 objectives) is used to count all the immune and inflammatory cells and any other type of nucleated cells employing random-field counting methodology until a total ofo400 cells have been counted. The count for each cell type is then expressed as a percentage of the total cells counted (differential percentage BAL cell count). For diagnostic purposes, all nucleated cells, not only inflammatory cells, must be included in the count to ensure that important information is not omitted (e.g. malignant cells, giant cells and epithelial cells). The presence of .5% bronchial epithelial cells indicates excessive contamination from the upper airways and such samples are inadequate as a reliable indicator of alveolar events.

Abnormal cell appearances must also be reported, including proportions of foamy macrophages, multinucleate macrophages, giant cells, macrophages containing smoking-related particles, macrophages containing refractile or birefringent particles indicative of inorganic dusts, or

macrophages heavily laden with haemosiderin confirmed by Perl staining, indicating possible pulmonary

haemosiderosis.

When neutrophil counts are very high it is important to check for intracellular bacteria, which can indicate active bacterial

pneumonia.

Fungal spores or hyphae may also be seen and their presence should be confirmed using Gomori–Grocott silver stain, which can also detectP. carinii.

Normal cell counts and the effect of smoking

BAL cells from healthy nonsmokers are mainly macrophages and a few lymphocytes but proportions of other cell types are very low.

Smoking causes increases in BAL

macrophages up to four-fold higher (total and per mL) in healthy smokers compared with nonsmokers; smokers also have slight increases in neutrophils. Thus, smoking must be taken into account when defining normal ranges and interpreting any BAL studies.

Published normal ranges show considerable variability when cell counts are expressed per mL or absolute total numbers. However, results are very similar when expressed as differential percentage counts, consistent with these not being influenced by dilution.

The normal ranges that can be employed for differential BAL cell counts are shown in table 2. Smoking-related inclusions are frequent in macrophages from smokers.

Main applications in the diagnostic work-up of peripheral lung diseases

Although this section describes BAL procedures, it would be incomplete without a summary of how BAL is used in routine clinical investigation to increase confidence in the diagnosis of many parenchymal lung diseases. A quick guide showing the main types of increased BAL inflammatory cells

and other cytological features in a wide range of lower respiratory diseases is given in table 1.