Folie 1

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Institute and Outpatient Clinic for Occupational
and Environmental Medicine Ziemssenstrasse 1
80336 Munich Germany http//arbmed.klinikum.uni-
muenchen.de
Non invasive methods used to assess occupational
exposures
Dennis Nowak, Rudolf Jörres
Institute and Outpatient Clnic for Occupational
and Environmental Medicine Ludwig-Maximilians-Univ
ersität München Germany
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Noninvasive monitoring of airway inflammation

• Sputum
• induced
• spontaneous
• Exhaled air
• Nitric oxide
• Carbon monoxide
• Breath condensate
• Electronic nose
• Temperature

• BHR / lung function
• Markers in serum / urine

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Induced sputum
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Induced sputum Cell differentials
Homogeneous distribution on cytospin slide
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Freezing of sputum samples for intermediate
storage
Eosinophil numbers not affected
Holz et al. CEA 2001
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Freezing of sputum samples Flow cytometry
CD3
CD4
frozen samples ( cells)
CD8
T4/T8
native sample ( cells)
Jaksztat et al. ERJ 2004
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Freezing of sputum samples for intermediate
storageis a valid procedure
15 DMSO -20C (normal freezing compartment)

• Eosinophils valid (cytospin)
• CD4/CD8 ratio valid (flow cytometry)

Holz et al. CEA 2001
Jaksztat et al. ERJ 2004
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Sputum cells indicate inflammatory response after
allergen challenge
Gauvreau et al. AJRCCM 1999
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Reproducibility of sputum analysis after allergen
challenge
Sputum eosinophils
7 h after challenge
24 h after challenge
100
100
()
()
80
80
60
60
second challenge
40
40
20
20
0
0
20
40
60
80
100
20
40
60
80
100
first challenge
first challenge
Gauvreau et al. JACI 1999
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Algorithm for diagnosis in patients with chronic
cough
History Clinical investigation X-ray
picture Spirometry Methacholine challenge
no clear diagnosis
Sputum induction
Jayaram et al. ERJ 2000
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Algorithm for diagnosis in patients with chronic
cough
Induced sputum
Eosinophilia
Neutrophilia elevated total cell number
Normal or Neutrophilia normal total cell number

• Eosinophilic bronchitiswithout asthma
• Occupational cause

• Infectious bronchitis(bakterial/viral/atypic)
• Bronchiectases
• Cystic fibrosis

• Gastrooesophagealreflux
• Habitual cough

Jayaram et al. ERJ 2000
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Sputum eosinophils and Occupational Asthma
(isocyanate)
Lemiere et al. ERJ 1999
Maestrelli et al. CEA 1994
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Sputum neutrophils/IL-8 and Occupational Asthma
(isocyanate)
8 patients with OA (7 patients with
corticosteroids)
Lemiere et al. CEA 2004
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Flow sheet for the diagnosis of Occupational
Asthma
49 patients with suspected OA
2 weeks at work (Peak flow)
induced sputum at work
2 weeks not at work (Peak flow)
induced sputum off work
specific challenge (SIC)
26 negative
23 positive
Girard et al. AJRCCM 2004
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Diagnosis of Occupational Asthma - Results
Girard et al. AJRCCM 2004
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Diagnosis of Occupational Asthma - Judgement by
experts
Sensitivity Specificity of diagnosis
(after specific challenge)
only peak flow peak flow/sputum

Girard et al. AJRCCM 2004
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Sputum neutrophils after Diesel exhaust exposure
FA
Diesel exhaust (PM10 300µg/m3)
IL-6
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Neutrophils
100
(pg/mL)
()
6 h after exposure
6 h after exposure
80

15
60

10
40
5
20
0
0
Healthy
Healthy
n 7
n 7
Nordenhall et al. ATS 2000
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WTC dust-exposed firefighters
10 months after September 11th, 2001
Comparison of 39 NY-FF, 12 TA-FF, 8 control
subjects (hospital workers)
Fireman et al. Environ Health Persp 2004
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WTC dust-exposed firefighters
Irregular particle formin WTC dust-exposure
Fireman et al.Environ Health Persp 2004
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WTC dust-exposed firefighters
Fireman, Environ Health Persp 2004
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Induced sputum evaluation in microwave popcorn
production workers
Exposure to popcorn flavoring agents (mainly
Deacetyl 2,3-butanedione)
Apkinar-Elci et al. Chest 2005
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Sputum induced cellularity in a group of traffic
policemen
Sputum neutrophils
plt0.01
Dragonieri et al. 2006
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Occupational eosinophilic bronchitis without
asthma due to chloramine (CLT) exposure
Time course of FEV1
Sputum analysis
Krakowiak et al. 2005
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Leukotrienes and isocyanate-induced asthma a
pilot study using induced sputum
Receptor expression on sputum neutrophils
Cysteinyl leukotriene (LTC4, LTD4) receptor
expression
BLT1 (mediating LTB4 response) receptor expression
Lemiere et al. CEA 2004
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Exhaled nitric oxide (eNO)
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Nitric oxide - physiological functions

• Vasodilation
• Host defence
• Neuronal transmission
• Inflammatory responses

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Cellular origin of eNO

• Epithelial endothelial cells
• Granulocytes
• Macrophages

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Exhaled nitric oxide (eNO) - Commercially
available analyzers
Aerocrine NIOX
Sievers NOA 280
EcoMedics CLD88
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Portable eNO device
NIOXMINO
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Exhaled nitric oxide (eNO)

• Possible sources

Nasal sinuses/nose extreme concentrations (gt1
ppm) ? exclude by measurement procedure (positive
pressure) Airways (bronchi) bronchoscopically
detectable derived from flow dependence of
concentration Alveoli high affinity of hemoglobin
to NO ? alveolar NO low, difficult to measure
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Measurement of exhaled nitric oxide
(eNO)-Procedure
Expiration, attach to mouthpiece
Deep inspiration (NO-free air)
Slow expiration against reistance control flow
rate by optical display or special resistance
Analyzer measures NO on-line by chemiluminescence.
3 measurements, values immediately available
Guidelines AJRCCM 2005
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Reproducibility of eNO measurements is high
2 measurements within about 2 weeks
Louw et al. DGP 2002
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Factors influencing eNO

• Smoking

• Spirometry

• Coffein

• Diet rich in nitrates

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eNO vs. BHR in asthma
Jatakanon et al. Thorax 1998
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Specific rise of eNO levels in patients
sensitized to latex 22 h after challenge
independent of asthma/rhinitis
sensitized nonsmokers
sensitized smokers
Baur et al. ERJ 2005
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Rise of eNO level after challengein patients
with west red cedar asthma
eNO associated with sputum eosinophils
Obata et al. ERJ 1999
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Effect of repeated peak ozone exposure on eNO as
marker of inflammation/asthma
Number of ozone peaks per year
Pulpmill workers
Olin et al. ERJ 2004
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Effect of an educational intervention in farmers
with occupational asthma on the level of exhaled
NO
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Effect of an educational intervention in farmers
with occupational asthma on the level of exhaled
NO
n.s.
n.s.
p0.009
p0.006
Subjects with FeNOgt35 ppb
All subjects
Dressel et al. ERS 2006
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Exhaled breath condensate (EBC)
Many compounds have been detected
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Exhaled breath condensate (EBC)
Collecting devices, two-way valve and cooler
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Hypothesis of aerosol generation in the lung
Generation of small droplets from the walls of
alveoli and small airways during the breathing
cycle
End of expiration
Inspiration
Mechanism different from coughing!
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Examples of compounds measured inexhaled air or
breath condensate

• NO
• CO
• Pentane, ethane ...
• Nitrite (NO2-), nitrate (NO3-)
• Nitrotyrosine
• Hydrogen peroxide (H2O2 )
• NH3/NH4
• Leukotrienes (LTB4, cysteinyl-LT)
• 8-Isoprostans
• Macromolecules (albumin, ECP...)
• pH, conductivity
• ..... ..... ... .. .

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Exhaled gases / breath condensate (EBC) vs
diagnosis

• NO ? Asthma, ? K.-S. (nasal)
• CO ? Asthma
• Pentane ? Asthma, ? ARDS
• NO2-, NO3- ? Asthma, ? CF
• Nitrotyrosine ? Asthma, ? CF
• H2O2 ? Asthma, ? COPD, ? CF, ? Bronchiectases, ?
  ARDS
• Leukotrienes ? Asthma
• 8-Isoprostane ? Asthma, ? COPD, ? CF, ?
  Sarcoidosis, ? ARDS, ? OSA
• NH3/NH4 ? Asthma, ? URTI
• pH ? Asthma, ? COPD, ? URTI, ? Bronchiectases

Specificity / Sensitivity not clarified
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H2O2 in breath condensate
Only short-time reproducibility
6 h
gt4 weeks
time between measurements
Holz et al. ATS 1999
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Analysis of exhaled air Further methods

• Carbon monoxide (CO) potential marker of
  oxidative stress
• Electronic nose alteration of hydrocarbon
  pattern in tumour patients
• Temperature profile of exhaled air bronchial
  blood flow

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Exhaled breath condensate (EBC)
Currently no clear-cut datain Occupational
Medicine
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