Download - R. Louis Pneumogie CHU Sart-Tilman, Liege LAnalyse des expectorations et du NO exhalé en Pneumolgie
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R. Louis
Pneumogie CHU Sart-Tilman, Liege
L’Analyse des expectorations et du NO exhalé en Pneumolgie
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Les expectorations
• Pathologies infectieuses
• Pathologies Tumorales
• Pathologies inflammatoires
• Pathologies de surcharge minérale
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Pathologies infectieuses
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Expectorations spontanées et infections respiratoires
“Expectorated sputum is among the least clinically relevant specimens received for culture in microbiology laboratories, even though it is one of the most numerous and time consuming specimens”
Bailley and Scott’s Diagnostic Microbiology, 1998
Echantillon obtenu après un effort de toux
Contamination minimale par la salive
Echantillon placé dans un container stérile
Transfert immédiat vers le laboratoire
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Critères d’acceptabilité d’un échantillon d’expectorations
Taux de cellules épithéliales squameuses < 10/low power field au microscope
Taux de cellules épithéliales squameuses < 10/low power field et taux de polynucléaires 25/low power field échantillon de qualité
Adéquat Inadéquat
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Expectorations (induites) et infections respiratoires basses
Pneumonie à Pneumocystis carinii
Mycobacterioses
Infections mycotiques
Légionelloses
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Pneumocystose
• 32 patients SIDA avec Rx Thorax anormale
• Induction d’expectorations avec NaCl 3%
• Fibroscopie avec BAL si pas de Pneumocystis dans expectorations
25 Cas de Pneumocystis Carinii dont 14 détecté par les expectorations sensibilité de 56%
Parmi les 18 patients sans Pneumocystis Carinii dans les expectorations 11 étaient positifs à l’analyse du BAL VPN de 39%
Bigby T et al Am Rev Respir Dis 1986
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Tuberculose pulmonaire
Sensibilité: 20 à 80%
VPP: 90%
Frotti d’expectorations coloré au Zielh-Neelsen
Macrophages (mauve) contenant le bacille tuberculeux (rouge)
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Aspergillose pulmonaire
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Pathologie tumorale
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Screening du cancer pulmonaire par analyse morphocytologique
NCI trial-Mayo Lung Project
Homme fumeur > 45 ans
Rx Thorax annuelle conseillée
Rx Thorax + cytologie des expectorations tous les 4 mois
Le nombre de Stade 1 et de cas chirurgicaux étaient plus grand dans le groupe avec screening régulier (respectivement 38% vs 25% et 46% vs 32%)
La survie à 5 ans est de 33%! comparée à 15% dans une population contrôle
Pas de différence en terme de mortalité totale due au cancer du poumon
Incidence de cancer plus grande dans le groupe avec screening régulier ??!!
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Contribution relative de l’analyse morphologique des cellules des expectorations vs RxThorax
dans le diagnostic du cancer
• Rx Thorax seule: 76%
• Expectorations seules: 17%
• Rx Thorax et expectorations: 7%
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Analyse des expectorations dans la détection du carcinome pulmonaire
L’analyse morphologique est surtout utile dans le carcinome épidermoïde où on peut diagnostiquer des cancers avec RxThorax normale (78 % au stade 1). Une cytologie positive pour d’autres types cellulaires est généralement une indication de stades avançés (NCI trial USA)
Des techniques “d’immunostaining” pour des antigènes fœtaux améliorent la sensibilité de l’analyse morphologique (hnRNP = heterogeneous nuclear ribonucleoprotein)
Détection de mutations géniques (K-ras oncogene ou p53) sur des cellules d’expectorations identiques à celle retrouvées dans le tissu cancereux 1 an avant le diagnostic clinique
Carcinome épidermoïde
Adénocarcinome
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Pathologie inflammatoire
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Induction des expectorations
Nébuliseur ultrasonique avec un débit de 1-3ml/min
Liquide hypertonique (3-5%)
Spiromètre ou peak flow meter
Prémédication du sujet avec 400 µg salbutamol (MDI+Spacer)
Temps d’inhalation de 10 à 20 min avec collecte toutes le 5 min d’un échantillon d’expectorations qui doivent être “poolés”
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Pour entire expectorate into preweighed polystyrene tube and
weigh
Add equal volume of DTT (DTE) 0.1%
Place in shaking waterbath or rocker for 15 min at 22°C
Filter through 48m nylon mesh
Perform manual TCC and assess viability
Centrifugation 1500 rpm 10 min at 4°C
Perform DCC on cytospins stained with GIEMSA(> 400 nonsquamous)
Select viscid or denser portion of the expectorate using an inverted microscope and place in preweighed polystyrene tube
Add DTT (DTE) 0.1% solution equivalent to 4 times the weight of the plug
Whole sputum Plug
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Differential sputum cells on cytospins
1
2
3 4
51. Macrophage
2. Neutrophil
3. Eosinophil
4. Epithelial cell
5. Lymphocytes
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Cytologie des expectorations induites
Cellule Pathologie
Eosinophiles
Neurophiles
Lymphocytes
Asthme
Bronchite chronique non tabagique
Exposition à des aéroallergènes ou des sensibilisants professionnels
BPCO sensible aux corticoïdes
Tabagisme
BPCO
Exposition aux endotoxines, à l’ozone
Asthme résistants aux corticoïdes
Infection
BBS
Chlamydia Pneumoniae
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Curschmann’s Spirals Charcot-Leyden cristal
Asthma
Chronic bronchitis
Asthma
Eosinophilic pneumonia
Fungal infection
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Severe asthma COPD
GERD Left ventricular dysfunction
100 µm
Lipid laden macrophages Haemosiderin laden macrophages
Massive eosinophiliaMassive neutrophilia
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Pin I et al Thorax 1992
Induced sputum in asthma
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Mild to moderate steroid naive asthma
Louis R et al Allergy 2002
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1
10
100
1000
10000
Eosin
op
hil
s (
X 1
03 /
g)
p<0.05
p<0.001
p<0.01
Control
group
Intermittent
asthma
Mild to moderate
asthma
Severe
asthma
p<0.001
Louis R et al Am J Respir Crit Care Med 2000
Sputum eosinophil counts in asthma
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70
80
90
100
110
0 5min
15min
30min
60min
2 h 3 h 4 h 5 h 6 h
FEV1
(% co
ntro
l)
Sal Dpt
0
5
10
15
20
25
30
35
40
Sput
um E
osin
ophi
ls (%
)6 h
Sal Dpt
Sputum eosinophil influx during a late phase bronchospasm
Bettiol J et al Allergy 2002
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Jatakanon A et al Am J Respir Crit Care Med 2000
Solid symbols: Exacerbation
Open symbols:
No exacerbation
Baseline sputum cell counts before tapering Inhaled corticosteroids
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0
2
4
6
8
10
12
14
16
Spu
tum
eos
inop
hils
(% c
hang
e)
No Exacerbation Exacerbations
Effect of exacerbation of asthma on changes in sputum eosinophils and FEV1
Jatakanon A et al Am J Respir Crit Care Med 2000
-30
-25
-20
-15
-10
-5
0
FE
V1
(% c
hang
e)
No Exacerbation Exacerbations
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Giannini D et al Clin Exp Allergy 2000
Sputum eosinophilia and withdrawal of inhaled steroids
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Proportion of mild to moderate asthmatics with raised sputum eosinophilia
Sputum eos < 2%
Sputum eos > 2%
69% 31%
Steroid naive patients
N = 118
Louis R et al Allergy 2002
< 2 >2-5 >5-10 >10-15>15-20 > 200
10
20
30
40
Pro
po
rtio
n o
f p
atie
nts
(%
)
Sputum Eosinophils (%)
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PC
20M
< 8
mg
/ml
Sp
utu
m e
os
> 1
%
FE
V1/
FV
C <
76.
6 %
BD
res
pons
e >
2.9
%
PE
FA
%M
> 2
1.6%
Blo
od e
os >
6.3
%
0
25
50
75
100
Ac
cu
rac
y (
%)
Accuracy of several tests to diagnose mild to moderate asthma
Hunter G et al Chest 2002
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< 3% > 3%
Age (yrs) 53 45
Atopy 2 8
FEV1( % pred) 81 86
PEF (amplitude % mean) 20 19
Symptom VAS (mm) 44 47
PC20 (mg/ml) 1.6 0.8
Sputum Eosinophils (%) 0.7 11
Non eosinophilic corticosteroid unresponsive asthma
Pavord I et al Lancet 1999
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-40
-30
-20
-10
0
10
20Eos > 3%
Eos < 3%p<0.05
NS
Ch
ang
e in
sym
pto
ms
VA
S (
mm
)
Pavord I et al Lancet 1999
Sputum eosinophilia as a predictive factor for response to inhaled corticoids in asthma
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-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0Eos > 3%Eos < 3%
p<0.05
NS
Ch
ang
e in
PC
20 (
DD
)
Pavord I et al Lancet 1999
Sputum eosinophilia as a predictive factor for response to inhaled corticoids in asthma
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ANTI-INFLAMMATORY TREATMENT:1.) Low dose inhaled steroid (100-200g BDP bd) 2.) Moderate dose inhaled steroid (200-400g BDP bd) 3.) High dose inhaled steroid (400-1600g BDP bd)4.) High dose inhaled steroid (400-1600g BDP bd) plus leukotriene antagonist 5.) Higher dose inhaled steroid (1600g BDP bd) plus leukotriene antagonist6.) Higher dose inhaled steroid (1600g BDP bd) plus leukotriene antagonist plus oral Prednisolone 30mg 2/52, then titrating dose reducing by 5mg/week
BRONCHODILATOR TREATMENT1.) Long acting 2 agonist2.) Long acting 2 agonist plus theophylline3.) Long acting 2-agonist plus theophylline plus nebulised bronchodilator
Step down anti-inflammatory treatment Step down bronchodilator
treatment
Step down anti-inflammatory treatment
Step up bronchodilator treatment SPUTUM EOSINOPHILS 1-
3%
Step up anti-inflammatory treatment
No change in bronchdilator treatment
No change in anti-inflammatory treatment
Step up bronchodilator treatment SPUTUM EOSINOPHILS
>3%
No change in anti-inflammatory Step down bronchodilator
treatment
Step up anti-inflammatory treatmentStep up bronchdilator treatment once
on maximum anti-inflammatory treatment
SPUTUM EOSINOPHILS <1%
Symptomscontrolled
Symptoms notcontrolled
Green et al. Lancet 2002
Sputum guided treatment adjustment
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BTS guidelines
sputum guidelines
120
100
80
60
40
20
0
Sev
ere
exac
erb
atio
ns
(cu
mu
lati
ve n
um
ber
)
0 1 2 3 4 5 6 987 10 11
Time (months)
12
‡p=0.01
1 asthma admission
6 asthma admissions
Green et al. Lancet Lancet 2002
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17051660Inh corticosteroids (mcg/day)
3.02.6Oral corticosteroids (mg/day)
1212Theophylline (n)
1513Leukotriene antagonists (n)
11*4Nebulised beta2 agonists (n)
1212Long acting beta2 agonists (n)
Traditional management
Sputum management
Treatment
*p<0.05 Green et al. Lancet in press
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Induced sputum in asthma
• Sputum eosinophilia is present in 70% of steroid naive asthmatics
• Measuring sputum eosinophil count together with methacholine bronchial responsiveness are the best ways to diagnose mild to moderate asthma
• Non eosinophilic asthmatics with prominent sputum neutrophilia seems to resist to inhaled corticoids
• Exacerbation of asthma is often associated with sputum eosinophilia
• Targeting sputum eosinophil count for adjusting the dose of inhaled corticoids in moderate to severe asthmatics help in reducing asthma exacerbation
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Sensitivity is lower in sputum than in BAL 75-100 % vs 25%
Specificity is higher in sputum > 95%
Asbestos body
Asbestose pulmonaire
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Le NO exhalé
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Le NO dans les voies aériennes
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7. Analyseur de NO (Monoxyde d’Azote)
Mesure « on line » par chemoluminescence au cours d’une expiration de 10 sec contre une résistance
Valeur moyenne normale :
21 ppb (part per billion)
Valeur significativement accrue:
> 45 ppb
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Exhaled NO in children
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Mesure du NO exhalé
• Flux expiratoire constant durant 10’’
• Débit expiratoire fixé à 50 ml/sec
• Contre une résistance
• Mesure durant les 4 dernières secondes
• 3 mesures réalisées et moyenne de ces 3 mesures
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Nasal vs bronchial NO
Karadag et al 1999
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Kharitonov S et al AJRCCM 2001
Raised ExNO in asthmatics
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Healthy subject Asthmatics
Epithelial activation as a source of exhaled NO in asthma
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Berlyne G et al JACI 2000
Performance of sputum eosinophil count vs exhaled NO for diagnosing asthma
Steroid naive patients Steroid treated patients
Solid line: sputum eosinophils
Dashed line: exhaled NO
Solid line: sputum eosinophils
Dashed line: exhaled NO
Cut-off: 0.3%
Cut-off: 17 ppb
Cut-off: 15 ppb
Cut-off: 0.08%
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Smith A et al Am J Respir Crit Care Med 2004
Performance of PEF variability, exNO and sputum eosinophil count to diagnose asthma
Solid line: NO (best cut-off 20 ppb)
Dashed line: Sputum Eosinophils (best cut-off 3%)
Solid line: variability in PEF
Dashed line: change in mean morning PEF after inhaled steroids
3%20 ppb
PEF
Sp Eo
Ex NO
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Use of exNO to guide treatment in asthma
Smith A N Engl J Med 2005
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Use of exNO to guide treatment in asthma
Smith A N Engl J Med 2005
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With the use of FeNO measurements, maintenance doses
of inhaled corticoids may be significantly reduced without compromising asthma control
Use of exNO to guide treatment in asthma
Smith A N Engl J Med 2005
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Nasal and exhaled NO in children
with PCD
Karadag et al 1999
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Factors affecting exhaled NO levels
+
-
Airway viral infection
Allergic rhinitis
Nitrate-rich diet
Spirometric maneuvres
Exercise
Alcohol
Bronchoconstriction
Ciliary dyskinesia
Hypertension
Smoking