A

newly

developed

tool

for

intra-tracheal

temperature

and

humidity

assessment

in

laryngectomized

individuals

the

Airway

Climate

Explorer

-LRB-

ACE

-RRB-

The

aim

of

this

study

is

to

develop

a

postlaryngectomy

airway

climate

explorer

-LRB-

ACE

-RRB-

for

assessment

of

intratracheal

temperature

and

humidity

and

of

influence

of

heat

and

moisture

exchangers

-LRB-

HMEs

-RRB-

Engineering

goals

were

within-device

condensation

prevention

and

fast

response

time

characteristics

The

ACE

consists

of

a

small

diameter

heated

air-sampling

catheter

connected

to

a

heated

sensor

house

containing

a

humidity

sensor

Air

is

sucked

through

the

catheter

by

a

controlled-flow

pump

Validation

was

performed

in

a

climate

chamber

using

a

calibrated

reference

sensor

and

in

a

two-flow

system

Additionally

the

analyser

was

tested

in

vivo

Over

the

clinically

relevant

range

of

humidity

values

-LRB-

mg

air

-RRB-

the

sensor

output

highly

correlates

with

the

reference

sensor

readings

-LRB-

-RRB-

The

response

times

are

all

s

A

first

in

vivo

pilot

measurement

was

successful

The

newly

developed

verified

fast-responding

ACE

is

suitable

for

postlaryngectomy

airway

climate

assessment

Introduction

Total

laryngectomy

results

in

a

permanent

disconnection

of

the

upper

and

lower

airways

and

leads

to

an

increase

in

chronic

pulmonary

complaints

like

frequent

involuntary

coughing

sputum

production

and

repeated

daily

forced

expectoration

in

order

to

clear

the

airways

-LSB-

-RSB-

Heat

and

moisture

exchangers

-LRB-

HMEs

-RRB-

were

developed

in

an

attempt

to

compensate

for

the

lost

functions

of

the

upper

airway

and

have

been

found

to

reduce

these

symptoms

and

improve

the

quality

of

life

-LSB-

-RSB-

In

vitro

the

moisture

exchanging

capacity

of

the

HME

can

be

specified

with

the

ISO

standard

However

this

standard

does

not

incorporate

a

standardized

method

for

determining

the

heat

exchange

of

the

HME

and

it

is

not

possible

to

translate

the

results

obtained

with

the

lung

model

of

the

ISO

standard

to

the

true

in

vivo

situation

Therefore

several

questions

remain

unanswered

Fundamentally

what

is

the

in

vivo

heat

and

moisture

exchanging

capacity

of

the

HME

And

clinically

how

does

the

in

vivo

heat

and

moisture

exchanging

capacity

relate

to

that

of

the

upper

airway

i.e.

where

does

the

HME

stand

in

the

range

from

open

stoma

to

nose

breathing

Is

a

further

improvement

of

the

HMEs

heat

and

moisture

exchanging

capacity

possible

and

desirable

and

does

this

result

in

a

further

decrease

of

the

pulmonary

complaints

due

to

the

use

of

this

rehabilitation

device

Measuring

intra

airway

temperature

and

humidity

is

a

technically

complex

issue

-LSB-

-RSB-

Assessing

the

temperature

and

humidity

variations

during

normal

breathing

requires

fast

sensor

response

characteristics

No

appropriate

commercial

systems

for

this

purpose

are

available

and

only

a

few

self-constructed

validated

systems

have

been

published

-LSB-

-RSB-

Two

groups

evaluated

the

influence

of

an

HME

on

intra

airway

temperature

and

humidity

in

laryngectomees

In

one

study

-LSB-

-RSB-

the

sensor

response

time

was

up

to

s

and

in

the

other

study

-LSB-

-RSB-

a

response

time

of

s

is

reported

which

has

been

shown

to

be

sufficiently

fast

for

normal

breathing

volunteers

but

which

might

be

insufficient

for

patients

Our

goal

was

to

develop

a

verified

intra-tracheal

airway

climate

analyser

with

response

characteristics

fast

enough

for

assessment

of

end

tidal

intra-airway

temperature

and

humidity

variations

during

normal

breathing

in

laryngectomized

individuals

and

for

evaluation

of

the

influence

of

HMEs

on

these

parameters

Design

considerations

Placement

of

a

humidity

sensor

in

the

airway

itself

was

rejected

Humidity

sensors

must

be

kept

clean

and

a

construction

to

protect

a

sensor

would

be

so

large

that

the

aerodynamics

within

the

airway

would

be

disturbed

A

high

airflow

around

the

sensor

is

required

to

minimize

the

response

time

of

the

sensor

A

constant

airflow

is

necessary

for

a

reliable

interpretation

of

the

time

dependent

signal

and

in

addition

facilitates

the

use

of

deconvolution

to

optimize

system

response

characteristics

Therefore

a

sensor

environment

had

to

be

created

in

which

airflow

remains

constant

and

high

during

the

whole

breathing

cycle

i.e.

in

a

chamber

In

order

to

disturb

airway

aerodynamics

as

little

as

possible

a

small

diameter

sample

catheter

for

transportation

of

breathing

air

outside

the

airway

to

a

humidity

sensor

is

required

We

decided

that

the

lumen

surface

of

the

sample

catheter

should

not

exceed

of

the

lumen

surface

of

the

trachea

A

decrease

in

lumen

area

of

maximal

will

result

by

the

law

of

mass

conservation

in

an

increase

in

local

flow

velocity

of

This

on

its

turn

will

lead

to

a

transbronchial

pressure

drop

of

maximal

which

is

supposed

to

be

small

enough

to

avoid

substantial

deformation

of

the

trachea

wall

This

means

that

based

on

a

diameter

of

cm

which

is

representative

for

the

trachea

of

an

adult

person

the

maximal

diameter

of

the

sample

catheter

should

not

be

more

than

mm

In

order

to

prevent

condensation

of

water

the

temperature

of

the

measurement

system

must

not

be

below

the

temperature

of

the

breathing

air

In

addition

high

humidity

levels

should

be

avoided

to

prevent

hysteresis

of

the

humidity

sensor

Based

on

the

maximal

i.e.

end

expiratory

temperature

of

about

°C

with

a

relative

humidity

-LRB-

RH

-RRB-

of

about

-LSB-

-RSB-

the

breathing

air

must

be

heated

to

at

least

°C

in

order

not

to

exceed

RH

-LRB-

-RRB-

On

the

other

hand

a

heated

sample

catheter

should

be

thermally

isolated

in

order

to

prevent

patient

discomfort

and

artificial

heating

of

inspired

air

We

decided

to

limit

the

sample

rate

to

less

than

of

the

respiratory

minute

volume

Based

on

a

breathing

frequency

of

at

least

cycles/minute

and

a

tidal

volume

of

l

for

an

adult

human

at

rest

the

sample

rate

should

be

l/min

at

most

Finally

the

catheter

should

be

easy

to

manipulate

and

it

should

be

possible

to

insert

it

into

the

trachea

even

if

an

HME

filter

is

in

situ

The

primary

measuring

site

for

the

sensor

should

be

about

cm

behind

the

HME

but

preferentially

measurements

deeper

in

the

trachea

should

be

possible

as

well

Components

Airway

Climate

Explorer

Breathing

air

is

sampled

through

a

mm

diameter

central

canal

of

a

multilumen

tube

-LRB-

see

Fig.

b.

-RRB-

Four

of

the

six

surrounding

canals

are

threaded

with

two

resistance

wire

loops

in

order

to

facilitate

catheter

heating

Resistance

wire

was

chosen

to

minimize

current

load

in

case

of

unexpected

patient

contact

Two

surrounding

canals

were

threaded

with

a

copper

wire

loop

in

order

to

measure

temperature

for

the

system

heating

control

unit

A

thermocouple

-LRB-

T-type

Ultra

Fast

Thermocouple

Probe

-LRB-

-RRB-

ADInstruments

Ltd

Oxfordshire

UK

-RRB-

was

placed

just

inside

the

tip

of

the

central

canal

in

order

to

establish

a

constant

airflow

around

this

sensor

The

thermal

response

of

the

thermocouple

is

specified

as

ms

accuracy

°C

The

distal

cm

of

the

sample

catheter

is

not

actively

heated

in

order

to

prevent

retrograde

heating

of

the

thermocouple

The

multilumen

tube

is

surrounded

by

a

silicone

isolation

tube

with

an

outer

diameter

of

mm

In

between

both

tubes

a

small

layer

of

air

increases

thermal

isolation

The

length

of

the

sample

catheter

is

cm

Fig.

of

the

sample

catheter

a

overview

with

enlargement

of

the

tip

b

cross

section

-LRB-

total

diameter

mm

-RRB-

The

proximal

end

of

the

catheter

is

connected

to

an

aluminium

sensor

house

incorporating

the

humidity

sensor

-LRB-

see

Fig.

-RRB-

The

humidity

sensor

most

suitable

for

our

purposes

appeared

to

be

the

capacitive

humidity

sensor

of

a

radiosonde

-LRB-

Vaisala

Oyj

Helsinki

Finland

-RRB-

The

technical

specifications

report

a

response

time

of

s

at

°C

and

s

at

°C

both

at

an

airflow

of

m/s

The

humidity

sensor

is

placed

in

an

aluminium

sensor

house

that

is

wrapped

with

copper

wire

for

heating

The

humidity

sensor

is

mounted

on

a

sensor

beam

and

consists

of

a

small

chip

with

the

sensor

polymer

and

an

aluminium

wire

that

in

the

original

radiosonde

served

as

a

heating

wire

but

which

we

use

to

measure

the

temperature

of

the

humidity

sensor

in

situ

-LRB-

see

Fig.

-RRB-

Apart

from

the

humidity

sensor

chip

the

sensor

beam

also

contains

a

temperature

sensor

but

this

was

not

used

as

it

is

located

relatively

far

from

the

humidity

sensor

chip

Fig.

section

of

the

sensor

house

I

connection

to

catheter

II

humidity

sensor

III

copper

wire

for

heating

of

the

sensor

house

IV

droplet

interceptor

with

electrode

for

fluid

detection

V

valve

for

disconnection

of

pressure

gradient

in

case

of

fluid

detection

in

the

droplet

interceptor

VI

thermocouple

wireFig

of

the

humidity

sensor

-LRB-

dimensions

-RRB-

The

sensor

house

is

heated

to

avoid

prolonged

saturation

of

the

sensor

-LSB-

-RSB-

The

temperature

is

kept

stable

within

°C

Therefore

the

output

of

the

capacitive

sensor

which

is

sensitive

for

the

relative

humidity

can

be

directly

converted

to

absolute

humidity

Because

a

radiosonde

is

not

designed

for

long-term

use

the

sensor

house

was

constructed

such

that

it

could

be

easily

opened

to

replace

the

sensor

beam

The

diameter

of

the

air

canal

that

leads

to

the

humidity

sensor

is

mm

The

air

inflow

passes

through

a

droplet

interceptor

in

the

sensor

house

in

order

to

prevent

sucked

up

sputum

from

being

projected

onto

the

sensor

chip

In

the

bottom

of

the

droplet

interceptor

an

electrode

is

placed

in

order

to

detect

fluid

accumulation

in

which

case

a

mechanic

valve

instantly

disconnects

the

pressure

gradient

through

catheter

and

sensor

house

which

is

established

by

a

vacuum

pump

-LRB-

DIVAC

Leybold

Vacuum

BV

Utrecht

the

Netherlands

-RRB-

The

flow

rate

is

controlled

and

kept

constant

at

l/min

by

a

mass

flow

meter/controller

-LRB-

Smart

Mass

Flow

Meter

type

and

Smart

Control

Software

Brooks

Instruments

Veenendaal

the

Netherlands

-RRB-

which

is

placed

between

the

sensor

house

and

the

pump

Figure

summarizes

the

airway

climate

explorer

in

a

block

diagram

In

this

figure

also

the

custom-made

electronic

module

is

shown

The

main

functions

of

the

module

are

processing

of

the

humidity

sensor

signal

and

its

associated

temperature

signal

i.e.

heating

of

both

the

sample

catheter

via

the

resistance

wire

and

the

sensor

house

via

the

copper

wire

each

to

their

own

adjustable

set

points

The

electronic

principles

of

the

design

of

the

module

are

described

in

the

appendix

Fig.

diagram

of

the

airway

climate

explorer

For

patient

safety

the

total

assembly

is

connected

to

a

safety-isolating

transformer

Data

acquisition

and

signal

processing

All

signals

of

the

total

assembly

i.e.

voltage

output

of

the

sensor

polymer

and

the

aluminium

wire

on

the

humidity

sensor

of

the

ACE

temperature

signal

of

the

thermocouple

sample

rate

of

the

mass

flow

controller

and

all

ancillary

equipment

-LRB-

see

below

-RRB-

are

read

out

simultaneously

via

a

multichannel

data

acquisition

system

with

additional

software

-LRB-

Powerlab

and

Chart

software

ADInstruments

Ltd

Oxfordshire

UK

-RRB-

on

a

PC

For

deconvolution

of

the

raw

humidity

output

of

the

ACE

the

software

application

LabVIEW

-LRB-

National

Instruments

Netherlands

BV

Woerden

The

Netherlands

-RRB-

is

used

Cleaning

and

disinfection

protocol

Prior

to

every

in

vivo

measurement

the

sample

catheter

is

cleaned

and

disinfected

with

the

following

procedure

First

the

catheter

is

rinsed

with

tap

water

Subsequently

the

catheter

is

cleaned

with

disinfection

hand

soap

-LRB-

Baktolin

Basic

Bode

Chemie

Hamburg

Germany

-RRB-

and

rinsed

again

with

tap

water

Then

the

catheter

is

placed

in

a

solution

of

Biotex

-LRB-

Sara

Lee

Utrecht

The

Netherlands

-RRB-

and

tap

water

for

min

after

which

it

is

placed

in

a

alcohol

water

solution

for

min

During

the

disinfection

procedure

chemicals

are

prevented

from

entering

the

central

canal

of

the

sample

catheter

by

establishing

retrograde

airflow

in

order

to

protect

the

humidity

sensor

polymer

The

central

canal

is

not

sterilized

because

during

use

there

is

continuous

suction

of

air

towards

the

vacuum

pump

Finally

the

catheter

is

dried

in

room

air

Ancillary

equipment

for

frequent

in

hospital

calibration

For

frequent

in-house

temperature

calibrations

a

can

of

water

is

used

in

which

the

thermocouple

is

placed

simultaneously

with

a

calibrated

thermometer

-LRB-

Thermalite

Electronic

Temperature

Instruments

Ltd

Worthing

UK

-RRB-

accuracy

°C

acting

as

a

secondary

reference

By

using

water

with

melting

ice

or

warm

tap

water

the

full

temperature

range

can

be

quickly

calibrated

For

frequent

humidity

calibrations

a

small

climate

room

was

constructed

This

climate

room

consists

of

a

plexiglas

box

of

in

which

moisturized

air

can

be

brought

via

the

distal

end

of

the

tube

of

a

neonatal

respiratory

humidifier

-LRB-

Respiratory

humidifier

Fisher

Paykel

Healthcare

Papendrecht

the

Netherlands

-RRB-

Air

is

mixed

via

an

electromotor-driven

propeller

-LRB-

cm

diameter

-RRB-

The

box

has

entries

for

the

tip

of

the

sample

catheter

and

for

a

calibrated

heated

humidity

and

temperature

sensor

accuracy

°C

and

relative

humidity

-LRB-

RH

-RRB-

-LRB-

Testo

B.V.

Almere

the

Netherlands

-RRB-

which

is

used

as

a

secondary

humidity

reference

The

system

can

be

operated

in

ambient

air

-LRB-

RH

-RRB-

can

be

connected

to

the

hospital

oxygen

supply

and

can

be

set

to

deliver

dry

or

humidified

gas

at

room

temperature

Assessment

of

step-response

characteristics

was

performed

in

a

two-stream

system

which

consisted

of

two

tubes

that

were

mounted

side

by

side

connected

to

in

absence

of

compressed

room

air

facilities

the

hospital

's

oxygen

supply

Through

one

tube

dry

oxygen

flowed

and

through

the

other

the

oxygen

was

heated

and

humidified

with

the

respiratory

humidifier

Subsequently

the

sample

catheter

tip

was

manually

switched

between

both

tube

lumina

Leakage

testing

is

performed

by

checking

the

time

required

to

completely

empty

a

flexible

sack

that

is

filled

with

a

well-defined

volume

of

air

-LRB-

l

-RRB-

This

volume

is

pushed

into

the

sack

with

a

calibration

syringe

-LRB-

Jaeger

GmbH/VIASYS

Healthcare

GmbH

Hoechberg

Germany

-RRB-

Environmental

temperature

and

humidity

are

monitored

with

the

heated

humidity

and

the

temperature

sensor

Calibration

and

verification

Full

calibration

of

the

system

was

performed

with

one

single

sensor

beam

However

due

to

the

disposable

design

of

a

radiosonde

and

the

possibility

of

damaging

the

sensor

during

the

measurements

most

likely

several

sensor

beams

will

be

used

over

time

Therefore

response

time

behaviour

was

checked

also

for

a

second

sensor

beam

and

the

calibration

procedure

was

designed

such

that

these

characteristics

can

be

easily

determined

for

a

consecutive

beam

Operating

temperature

Raising

the

temperatures

of

the

system

and

therefore

of

the

humidity

sensor

above

room

temperature

we

observed

an

initial

decrease

in

the

response

time

but

above

°C

the

response

time

increased

Therefore

°C

was

chosen

as

the

operating

temperature

At

this

temperature

the

sampled

air

in

the

catheter

reaches

°C

well

before

arrival

in

the

sensor

house

and

therefore

the

influence

of

breathing

temperature

variations

on

sensor

temperature

is

negligible

For

measurements

at

cm

deep

in

the

trachea

the

heated

part

of

the

tube

does

not

enter

the

trachea

so

that

artificial

heating

of

the

inspired

air

will

not

occur

Static

calibration

The

temperature

of

the

sensor

is

kept

stable

within

°C

Therefore

the

output

of

the

capacitive

sensor

which

is

sensitive

for

the

relative

humidity

can

be

directly

converted

to

absolute

humidity

In

a

climate

cabinet

-LRB-

Heraeus

Votsch

-RRB-

the

humidity

output

of

the

ACE

was

plotted

against

a

clinically

relevant

range

of

absolute

humidity

values

as

measured

with

the

secondary

reference

sensor

see

Fig.

Since

this

relation

appeared

to

be

linear

a

calibration

is

considered

appropriate

for

further

frequent

humidity

calibrations

in

the

hospital

Given

the

accuracy

of

the

reference

sensor

and

the

somewhat

limited

range

between

the

calibration

points

to

avoid

excessive

condensation

or

temperature

disequilibrium

in

the

calibration

device

the

inaccuracy

of

the

system

for

measuring

absolute

humidity

values

after

the

calibration

is

less

than

Fig.

of

the

humidity

sensor

plotted

against

the

secondary

reference

The

thermocouple

measuring

the

temperature

at

the

tip

of

the

sample

catheter

was

calibrated

against

the

values

of

the

reference

thermometer

in

ice

water

and

water

at

body

temperature

This

procedure

can

be

performed

in

the

hospital

prior

to

every

in

vivo

measurement

Given

the

accuracy

of

the

reference

thermometer

the

accuracy

of

the

system

for

measuring

temperature

values

after

a

calibration

is

°C

Assessing

the

role

of

condensation

in

the

measuring

system

In

order

to

assess

the

necessity

of

heating

the

system

in

order

to

prevent

condense

formation

within

the

device

step

response

measurements

were

performed

with

an

unheated

system

and

subsequently

with

the

system

heated

to

°C

With

an

unheated

system

a

delay

of

up

to

several

seconds

was

observed

in

the

humidity

trace

relative

to

the

thermocouple

trace

after

a

downward

step

The

longer

the

sensor

is

kept

in

a

high

humidity

environment

this

delay

increases

and

it

is

indicative

of

the

formation

and

subsequent

evaporation

of

the

condensate

within

the

system

Even

when

simulating

clinically

relevant

breathing

frequencies

a

noticeable

delay

of

s

was

observed

When

measuring

high

humidity

at

operating

temperature

for

a

long

time

after

the

downward

step

a

very

fast

response

with

a

delay

of

less

than

s

was

observed

This

delay

is

caused

by

the

transport

of

sampled

air

from

the

tip

of

the

catheter

towards

the

humidity

sensor

and

is

not

relevant

for

the

measurement

of

end

tidal

values

In

the

downward

trace

this

fast

response

was

followed

by

a

shoulder

at

about

of

the

step

of

which

the

length

again

increased

with

the

duration

at

high

humidity

This

shoulder

probably

represents

the

evaporation

of

the

condensate

in

the

unheated

tip

of

the

sensor

During

in

vivo

measurements

however

the

system

will

never

be

exposed

to

high

humidities

for

an

extended

period

of

time

Therefore

we

simulated

a

range

of

breathing

frequencies

with

the

two-stream

system

resulting

in

a

series

of

block

functions

of

approximately

and

cpm

-LRB-

cycles

per

minute

-RRB-

At

cpm

a

short

shoulder

is

still

visible

but

the

condensate

evaporates

sufficiently

fast

that

the

end-inspiratory

value

is

reached

At

and

cpm

no

shoulder

was

observed

Raw

response

time

characteristics

Using

the

two-stream

system

the

step

response

of

the

ACE

was

determined

Both

for

the

temperature

and

the

humidity

signal

we

determined

the

response

time

For

the

downward

humidity

step

we

could

not

use

a

plain

step

function

because

condensation

in

the

tip

would

occur

Therefore

we

used

a

block

function

with

a

frequency

of

cpm

-LRB-

see

Fig.

-RRB-

At

this

frequency

the

response

time

is

still

relatively

short

compared

with

the

period

of

the

block

function

and

easy

to

determine

from

the

humidity

trace

Fig.

to

a

block

function

solid

black

line

raw

humidity

trace

solid

grey

line

deconvolved

humidity

trace

dashed

line

thermocouple

trace

Vertical

axis

°C

mg

Horizontal

axis

seconds

a

Response

to

a

block

function

of

approximately

cycles

per

minute

b

Overview

of

the

response

to

a

block

function

with

varying

frequency

-LRB-

to

cpm

-RRB-

For

the

thermocouple

the

response

to

increasing

and

decreasing

temperature

steps

was

s.

For

the

first

humidity

sensor

the

response

to

increasing

humidity

and

decreasing

humidity

was

and

s

respectively

at

a

frequency

of

cpm

We

verified

that

the

response

characteristics

at

other

frequencies

were

comparable

Both

rise

and

fall

times

of

the

second

humidity

sensor

were

approximately

shorter

The

observed

raw

response

times

are

slightly

longer

than

we

had

expected

based

on

the

specifications

of

the

Vaisala

Humidity

Sensor

probably

because

the

airflow

over

the

sensor

is

slower

than

specified

Although

every

attempt

was

made

to

minimize

the

volume

of

the

sensor

chamber

we

estimate

that

the

airspeed

over

the

sensor

is

between

and

m/s

instead

of

m/s

Deconvolution

of

the

humidity

signal

Since

the

raw

step

response

times

of

tested

humidity

sensors

were

considered

too

slow

for

patients

with

high

breathing

frequencies

it

was

decided

to

further

process

the

raw

humidity

signal

Depending

on

the

tested

sensor

the

raw

step

response

can

be

closely

approximated

by

a

single

or

a

double

exponential

function

Therefore

it

is

possible

to

reconstruct

the

original

signal

by

deconvolution

Although

we

observed

a

different

response

time

for

increasing

and

decreasing

humidity

steps

we

nevertheless

chose

a

single

function

because

of

its

better

numerical

stability

For

the

removal

of

excessive

noise

a

low

pass

filter

of

cpm

was

applied

Because

the

raw

response

characteristics

varied

in

between

the

humidity

sensors

and

over

time

τ

was

adjusted

for

each

individual

measurement

The

principle

of

adjustment

of

these

parameters

was

to

acquire

a

maximal

value

for

τ

such

that

no

overshoot

of

the

deconvolved

humidity

trace

was

visible

We

used

the

thermocouple

signal

as

reference

for

the

true

step

function

For

the

first

sensor

the

deconvolved

signal

was

stable

and

optimal

for

increasing

humidity

with

τ

s.

For

the

second

humidity

sensor

a

τ

of

s

appeared

to

be

appropriate

We

verified

that

the

raw

step

response

characteristics

were

reproducible

and

that

the

deconvolution

results

were

not

influenced

by

the

step

size

After

deconvolution

of

the

humidity

signal

the

response

characteristics

improved

see

Fig.

The

step

response

time

to

a

block

function

for

the

first

humidity

sensor

was

s

for

increasing

humidity

and

s

for

decreasing

humidity

at

a

frequency

of

cpm

For

the

second

sensor

the

deconvolved

step

response

characteristics

were

similar

after

deconvolution

Figure

shows

the

raw

and

deconvolved

humidity

response

to

a

block

function

over

a

range

of

breathing

frequencies

It

is

shown

that

the

frequency

dependence

of

the

peak-to-peak

values

is

reduced

after

deconvolution

At

higher

frequencies

we

nevertheless

see

that

the

deconvolved

signal

increasingly

deviates

from

the

true

peak-to-peak

values

This

deviation

is

more

marked

in

the

lower

peak

due

to

the

relatively

long

humidity

fall

time

of

the

raw

trace

compared

to

the

rise

time

The

magnitude

of

the

deviation

in

the

lower

peak

ranges

from

about

mg

at

a

breathing

frequency

of

cpm

to

mg

at

cpm

Since

the

step

response

characteristics

of

the

deconvolved

signal

were

comparable

for

both

tested

sensors

the

deviations

are

of

comparable

magnitude

too

At

cpm

even

the

amplitude

of

the

fast

thermocouple

signal

does

not

reach

the

true

peak-to-peak

values

The

deviation

is

less

than

°C

so

that

we

do

not

consider

a

correction

necessary

for

the

thermocouple

signal

The

in

vivo

measurement

The

total

assembly

and

the

influence

of

a

heat

and

moisture

exchanger

were

tested

in

one

laryngectomized

volunteer

The

tip

of

the

sample

catheter

was

pushed

cm

through

a

small

hole

that

was

punched

in

an

HME

plaster

-LRB-

Fig.

-RRB-

The

diameter

of

the

puncture

was

slightly

smaller

than

the

diameter

of

the

sample

catheter

in

order

to

avoid

air

leakage

Figure

shows

a

segment

of

the

raw

and

deconvolved

humidity

trace

and

the

thermocouple

signal

In

this

particular

volunteer

τ

required

adjustment

to

in

order

to

eliminate

overshoot

During

breathing

in

rest

intra

airway

temperature

and

humidity

ranged

from

°C

and

mg

-LRB-

typical

end

inspiratory/end

expiratory

values

-RRB-

respectively

During

the

total

measurement

session

breathing

frequency

ranged

from

to

cpm

-LRB-

mean

cpm

SD

cpm

-RRB-

In

addition

inspiration

time

may

be

relatively

short

compared

to

expiration

time

See

the

beginning

of

the

trace

in

Fig.

Fig.

catheter

in

situ

pushed

through

a

punched

hole

in

the

HME

plaster

without

HME

filter

in

situFig

vivo

humidity

trace

before

and

after

deconvolution

black

trace

raw

humidity

signal

grey

trace

deconvolved

humidity

signal

Vertical

axis

mg

Horizontal

axis

seconds

Discussion

Our

results

show

that

we

have

succeeded

in

developing

an

intra-tracheal

airway

climate

analyser

with

response

characteristics

fast

enough

for

assessment

of

end

tidal

intra

airway

temperature

and

humidity

variations

during

normal

breathing

in

laryngectomized

individuals

In

the

literature

only

a

few

systems

have

been

described

that

simultaneously

measure

temperature

and

humidity

in

the

human

airway

-LSB-

-RSB-

Ingelstedt

-LSB-

-RSB-

reported

on

intra

airway

climate

in

healthy

individuals

using

a

well

validated

system

based

on

psychrometry

with

signal

stability

s

after

sudden

exposure

to

increased

humidity

However

this

method

had

a

few

disadvantages

The

wet

thermo

element

remained

sufficiently

moistened

for

only

respiratory

cycles

after

which

it

had

to

be

retracted

in

order

to

be

watered

by

a

moistened

wick

again

Furthermore

even

the

slightest

contact

with

tracheal

secretion

increased

the

thermal

inertia

of

the

psychrometer

after

which

it

has

to

be

cleaned

in

a

lasting

procedure

Although

contact

of

the

humidity

sensor

of

the

ACE

by

tracheal

secretion

is

less

likely

the

central

canal

of

the

sample

catheter

is

susceptible

to

obstruction

by

tracheal

secretion

When

this

would

occur

the

sample

catheter

can

be

disconnected

from

the

sensor

house

and

subsequently

the

obstructive

plug

can

be

removed

by

establishing

reversed

airflow

The

electrodes

in

the

bottom

of

the

droplet

interceptor

appeared

to

work

effectively

If

the

humidity

sensor

would

be

damaged

it

can

easily

be

replaced

Due

to

the

in

hospital

calibration

procedures

the

measurements

can

be

continued

within

a

relatively

short

time

span

The

system

of

Primiano

et

al.

-LSB-

-RSB-

consisted

of

a

sample

catheter

that

was

connected

to

a

mass

spectrometer

In

order

to

prevent

condensation

the

tip

of

their

sample

catheter

was

constricted

establishing

a

decrease

in

air

pressure

Extensive

validation

of

the

total

assembly

revealed

impressive

response

time

characteristics

of

less

that

s

for

humidity

readings

and

less

than

s

for

temperature

readings

respectively

Since

mass

flow

meters

are

expensive

and

require

solid

maintenance

the

use

of

such

a

device

was

not

considered

feasible

for

our

project

Although

their

measurement

system

has

been

used

for

assessment

of

anaesthesiology

heat

and

moisture

exchanger

effectiveness

we

are

not

aware

that

this

system

has

been

used

for

airway

climate

assessment

in

laryngectomees

McRae

et

al.

-LSB-

-RSB-

and

Keck

et

al.

-LSB-

-RSB-

have

assessed

tracheal

climate

and

the

influence

of

HMEs

in

laryngectomees

The

set

up

of

Keck

et

al.

consisted

of

a

sample

catheter

connected

to

a

sensor

house

that

incorporated

a

capacitive

humidity

sensor

As

described

in

-LSB-

-RSB-

the

sensor

house

was

heated

with

a

heat

mat

to

°C

which

is

slightly

lower

than

the

maximum

end

expiratory

temperature

observed

-LRB-

°C

-RRB-

so

that

condensation

still

might

be

a

possibility

However

the

sample

catheter

was

not

heated

and

the

verification

of

the

total

assembly

has

not

been

included

in

the

report

In

the

present

device

evidence

of

condensation

probably

precipitated

in

the

unheated

distal

cm

of

the

catheter

tip

-LRB-

which

is

in

vivo

somewhat

heated

by

the

body

-RRB-

is

observed

when

offering

block

functions

at

a

frequency

of

cpm

Despite

this

at

cpm

the

end

values

were

not

affected

It

is

therefore

unlikely

that

also

during

in

vivo

measurements

condensation

will

affect

the

amplitude

of

the

peak-to-peak

values

Moreover

the

temperature

and

humidity

steps

will

occur

more

gradually

in

the

in

vivo

situation

theoretically

decreasing

the

amount

of

condense

formation

in

the

sensor

tip

compared

to

the

offered

block

function

In

the

studies

by

McRae

et

al.

-LSB-

-RSB-

the

response

time

of

the

humidity

sensor

used

in

the

setup

of

McRae

was

up

to

s

The

frequency

of

human

rest

breathing

is

far

too

high

compared

to

these

response

characteristics

to

obtain

representative

values

for

intra-tracheal

humidity

However

as

the

two-stream

block

function

experiment

has

shown

also

in

the

present

measuring

device

the

amplitude

of

the

deconvolved

humidity

trace

depends

on

the

breathing

frequency

The

clinical

implication

of

this

finding

is

that

at

higher

breathing

frequencies

the

true

peak-to-peak

values

are

slightly

but

progressively

underestimated

by

the

ACE

although

probably

less

than

in

vitro

due

to

more

gradual

humidity

and

temperature

variation

in

the

latter

situation

The

variation

in

end-inspiratory

humidity

values

and

temperature

signals

as

indicated

by

the

deconvolved

humidity

signal

and

the

thermocouple

in

the

in-vivo

experiment

may

therefore

be

partially

due

to

a

frequency

dependency

and

not

due

to

a

real

variation

in

humidity

and

temperature

values

Quality

control

The

first

sensor

functioned

correctly

for

year

and

the

second

for

half

a

year

Both

sensors

had

to

be

replaced

because

of

accidental

damage

during

cleaning

The

two

humidity

sensors

appeared

to

have

different

response

characteristics

Sensor

age

variation

in

the

production

process

of

the

humidity

sensor

etc.

may

result

in

variation

between

the

characteristics

of

different

humidity

sensors

but

also

between

the

characteristics

of

one

sensor

measured

over

time

Linearity

between

reference

humidity

values

and

sensor

output

in

all

consecutive

sensors

is

felt

to

be

a

prerequisite

for

serial

production

of

radiosondes

The

small

climate

room

and

the

calibration

is

therefore

considered

appropriate

for

daily

quality

control

A

potential

source

of

error

is

leakage

of

air

causing

dilution

of

sampled

tracheal

air

with

room

air

Therefore

the

simple

leakage

test

should

be

part

of

the

regular

quality

control

Although

both

tested

humidity

sensors

revealed

the

different

step

response

times

of

the

raw

humidity

signal

the

peak-to-peak

amplitude

of

deconvolved

signal

was

comparable

between

both

sensors

The

two-stream

step

response

set

up

facilitates

regular

control

of

the

sensor

response

characteristics

and

if

necessary

adjustment

of

the

deconvolution

function

can

be

easily

performed

Limitations

and

further

improvements

During

measurements

deeper

in

the

trachea

-LRB-

e.g.

cm

-RRB-

contact

of

the

catheter

tip

with

the

airway

wall

may

lead

to

irresistible

cough

Preferably

the

distal

end

of

the

sample

catheter

should

be

better

pliable

in

the

shape

of

the

trachea

in

order

to

facilitate

better

control

of

its

tip

and

reducing

airway

wall

contact

Also

visual

control

of

the

catheter

tip

may

contribute

to

prevention

of

wall

contact

The

response

of

the

sensor

when

decreasing

the

humidity

is

slower

than

the

response

to

increasing

humidity

However

the

same

simple

deconvolution

function

is

used

for

increasing

and

decreasing

humidity

A

more

advanced

signal

processing

might

further

improve

response

characteristics

and

decrease

the

remaining

frequency

dependence

of

the

peak

tot

peak

amplitude

In

particular

for

the

end-inspiratory

values

this

is

important

because

of

the

relatively

short

time

span

of

the

inspiration

time

compared

to

the

expiration

time

combined

with

the

relatively

slow

response

characteristics

to

decreased

humidity

compared

to

increased

humidity

In

conclusion

the

Airway

Climate

Explorer

is

an

easy

to

use

relatively

inexpensive

tool

for

intra

airway

temperature

and

humidity

measurements

When

looking

at

the

end-inspiratory

and

end-expiratory

humidity

values

as

measured

by

our

system

the

accuracy

is

adequate

for

assessment

of

intratracheal

climate

O O O O O O B O B O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O B O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O B I O O O O O O O O O O O O O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O O

heat and moisture exchanger
airway climate
temperature
humidity


DNA

Microarrays

for

Identifying

Fishes

In

many

cases

marine

organisms

and

especially

their

diverse

developmental

stages

are

difficult

to

identify

by

morphological

characters

DNA-based

identification

methods

offer

an

analytically

powerful

addition

or

even

an

alternative

In

this

study

a

DNA

microarray

has

been

developed

to

be

able

to

investigate

its

potential

as

a

tool

for

the

identification

of

fish

species

from

European

seas

based

on

mitochondrial

rDNA

sequences

Eleven

commercially

important

fish

species

were

selected

for

a

first

prototype

Oligonucleotide

probes

were

designed

based

on

the

rDNA

sequences

obtained

from

individuals

of

fish

species

In

addition

more

than

sequences

of

species

served

as

sequence

background

against

which

the

specificity

of

the

probes

was

tested

in

silico

Single

target

hybridisations

with

PCR-amplified

rDNA

fragments

from

each

of

the

species

on

microarrays

containing

the

complete

set

of

probes

confirmed

their

suitability

True-positive

fluorescence

signals

obtained

were

at

least

one

order

of

magnitude

stronger

than

false-positive

cross-hybridisations

Single

nontarget

hybridisations

resulted

in

cross-hybridisation

signals

at

approximately

of

the

cases

tested

but

all

of

them

were

at

least

one

order

of

magnitude

lower

than

true-positive

signals

This

study

demonstrates

that

the

rDNA

gene

is

suitable

for

designing

oligonucleotide

probes

which

can

be

used

to

differentiate

fish

species

These

data

are

a

solid

basis

for

the

second

step

to

create

a

Fish

Chip

for

approximately

fish

species

relevant

in

marine

environmental

and

fisheries

research

as

well

as

control

of

fisheries

products

Introduction

Compared

with

terrestrial

ecosystems

little

is

known

about

marine

biodiversity

and

changes

in

species

richness

and

ecosystem

function

This

is

mainly

because

of

sampling

difficulties

and

problems

in

taxonomy

Many

marine

organisms

and

especially

their

diverse

developmental

stages

such

as

-LRB-

-RRB-

eggs

and

larvae

of

fishes

and

invertebrates

-LRB-

-RRB-

zoo

and

phytoplankton

and

-LRB-

-RRB-

benthic

invertebrates

are

cumbersome

and

difficult

to

identify

by

morphological

characters

Classical

microscopy

methods

are

time-consuming

and

require

a

high

degree

of

taxonomic

expertise

which

is

currently

falling

short

In

many

cases

identifying

a

species

is

the

major

bottleneck

in

marine

biodiversity

and

ecosystem

research

hampering

the

necessary

monitoring

of

marine

biodiversity

As

an

example

a

review

of

studies

on

invertebrate

diversity

in

European

seas

showed

that

approximately

one-third

of

the

specimens

could

not

have

been

identified

to

species

level

-LRB-

Schander

and

Willassen

-RRB-

DNA-based

identification

methods

are

meanwhile

established

-LRB-

Barlow

and

Tzotzos

-RRB-

as

powerful

tools

exhibiting

an

unprecedented

accuracy

because

of

their

inherently

highest

possible

resolution

which

can

reach

even

the

level

of

single

base

changes

in

a

whole

genome

Using

these

methods

the

following

marine

animals

have

been

investigated

eggs

larvae

and

adults

of

fishes

-LRB-

Rocha-Olivares

Noell

et

al.

Fox

et

al.

Ward

et

al.

-RRB-

planktonic

copepods

-LRB-

Bucklin

et

al.

-RRB-

invertebrate

larvae

-LRB-

Garland

and

Zimmer

Barber

and

Boyce

-RRB-

and

prey

in

the

gut

content

or

feces

of

penguins

whales

and

fishes

-LRB-

Jarman

et

al.

Saitho

et

al.

-RRB-

Sequences

of

the

small

subunit

of

the

rRNA

gene

are

used

as

a

standard

method

for

identifying

microbial

organisms

-LRB-

Ludwig

et

al.

-RRB-

and

a

fragment

of

the

mitochondrial

cytochrome

oxidase

I

gene

is

in

use

as

DNA-barcode

for

the

identification

of

metazoans

-LRB-

Hebert

et

al.

Ward

et

al.

-RRB-

As

shown

above

a

growing

number

of

recently

published

studies

are

using

molecular

genetic

identification

methods

Nevertheless

their

application

is

still

restricted

mainly

because

of

methodical

problems

and

because

special

knowledge

and

experience

in

molecular

genetics

is

required

This

is

especially

true

if

DNA-based

identification

is

performed

by

using

microarray

platforms

that

are

error-prone

and

difficult

to

quantify

-LRB-

Shi

et

al.

-RRB-

Whereas

most

of

the

methods

presently

in

use

such

as

PCR-based

DNA

amplification

followed

by

sequencing

techniques

allow

to

handle

only

single

or

a

few

species

at

the

same

time

DNA

microarrays

are

believed

to

have

the

potential

of

identifying

hundreds

of

species

in

parallel

and

to

differentiate

them

against

an

even

larger

number

of

related

species

Commonly

microarrays

are

glass

microscope

slides

on

which

oligonucleotide

probes

are

spotted

that

are

complementary

to

the

DNA

target

sequences

to

be

analysed

-LRB-

Relógio

et

al.

Pirrung

Dufva

-RRB-

The

DNA

target

which

is

usually

fluorophore-labelled

during

PCR

amplification

hybridises

with

the

oligonucleotide

probe

on

the

microarray

and

can

be

detected

after

washing

steps

by

its

label

Whereas

applying

of

DNA

microarrays

for

gene

expression

has

already

reached

the

routine

level

of

high

throughput

systems

-LRB-

Blohm

and

Guiseppi-Elie

Hoheisel

-RRB-

they

have

been

only

recently

applied

for

the

identification

of

organisms

such

as

microbes

-LRB-

Wang

et

al.

Call

et

al.

Korimbocus

et

al.

Loy

and

Bodrossy

-RRB-

animals

-LRB-

Pfunder

et

al.

-RRB-

and

plants

-LRB-

Rønning

et

al.

-RRB-

In

terms

of

identifying

marine

organisms

microarrays

have

been

used

for

bacteria

-LRB-

Peplies

et

al.

Peplies

et

al.

-RRB-

and

phytoplankton

-LRB-

Metfies

and

Medlin

Metfies

et

al.

Godhe

et

al.

-RRB-

Other

DNA-hybridisation

methods

for

the

identification

of

higher

marine

organisms

such

as

invertebrate

larvae

-LRB-

Goffredi

et

al.

-RRB-

copepods

-LRB-

Kiesling

et

al.

-RRB-

and

larvae

of

fishes

-LRB-

Rosel

and

Kocher

-RRB-

have

been

applied

but

microarrays

have

not

been

used

for

this

kind

of

studies

Other

applications

of

microarrays

in

research

on

marine

organisms

are

gene

expression

analysis

-LRB-

Williams

et

al.

Lidie

et

al.

Wang

et

al.

Cohen

et

al.

Jenny

et

al.

-RRB-

and

genotyping

in

population

genetics

-LRB-

Moriya

et

al.

Moriya

et

al.

-RRB-

One

of

the

methodical

limitations

for

using

microarrays

is

the

design

of

species

specific

probes

On

the

one

hand

oligonucleotide

probes

designed

in

silico

do

not

always

exhibit

the

experimental

hybridisation

properties

they

were

selected

for

and

must

be

empirically

tested

On

the

other

hand

the

molecular

marker

must

have

highly

selective

characteristics

such

as

low

intraspecific

and

a

high

interspecific

variation

One

of

the

most

frequently

used

markers

in

phylogenetics

of

fishes

is

the

mitochondrial

rRNA

gene

This

gene

has

a

well-characterized

secondary

structure

-LRB-

Meyer

Ortí

et

al.

-RRB-

and

especially

the

loop

regions

exhibit

many

insertions

deletions

and

substitutions

forming

highly

variable

molecular

features

which

usually

allow

the

design

of

highly

specific

probes

This

is

underlined

by

a

study

on

lionfishes

-LRB-

Kochzius

et

al.

-RRB-

which

showed

that

individuals

of

one

species

exhibit

identical

rDNA

haplotypes

even

though

they

were

sampled

at

sites

thousands

of

kilometres

apart

but

clear

differences

could

be

detected

between

closely

related

lionfish

species

In

this

study

the

development

of

a

DNA

microarray

is

described

demonstrating

the

suitability

of

the

rRNA

gene

for

designing

oligonucleotides

as

microarray

probes

to

differentiate

at

least

fish

species

from

European

seas

Based

on

these

data

a

Fish

Chip

for

approximately

fish

species

is

under

construction

to

support

the

identification

of

eggs

and

larval

stages

from

species

that

are

otherwise

difficult

to

identify

and

of

adult

or

processed

fishes

in

fisheries

industry

Material

and

Methods

Sampling

and

DNA

Extraction

To

consider

possible

intraspecies

sequence

variations

fishes

were

collected

in

five

different

regions

of

the

European

seas

North

Sea

Bay

of

Biscay

and

Western

Central

as

well

as

Eastern

Mediterranean

-LRB-

Fig.

Table

-RRB-

All

together

individual

samples

from

fish

species

were

investigated

In

addition

sequences

from

the

EMBL

sequence

data

base

were

included

Voucher

specimens

and

tissue

samples

have

been

preserved

in

absolute

ethanol

and

stored

at

°C

or

were

frozen

at

°C

DNA

was

extracted

from

gill

filaments

with

the

Agowa

mag

midi

DNA

isolation

kit

-LRB-

AGOWA

Berlin

Germany

-RRB-

or

from

muscle

tissue

with

the

DNeasy

tissue

kit

-LRB-

Qiagen

Hilden

Germany

-RRB-

according

to

the

instructions

of

the

manufacturer

Figure

with

the

sampling

areas

NS

North

Sea

BB

Bay

of

Biscay

WM

Western

Mediterranean

CM

Central

Mediterranean

EM

Eastern

Mediterranean.Table

of

sequences

per

fish

species

and

sampling

regionSpeciesFamilyOrderNorth

SeaBay

of

BiscayWestern

MediterraneanCentral

MediterraneanEastern

MediterraneanEMBL

sequence

data

base

and

other

projectsTotalAccession

number

of

EMBL

data

base

sequencesTarget

species

Boops

Engraulis

Helicolenus

Lophius

Pagellus

Scomber

Scophthalmus

Serranus

Sparus

Trachurus

Trigla

species

Chelidonichthys

Diplodus

Gadus

Merluccius

Mullus

Mullus

Pagellus

Platichthys

Pleuronectes

Psetta

Sardina

Scorpaena

Scorpaena

Serranus

Solea

Zeus

Σ

numbers

are

given

for

sequences

obtained

from

EMBL

sequence

databaseTaxonomy

according

to

FishBase

-LRB-

-RRB-

Polymerase

Chain

Reaction

and

Sequencing

A

fragment

of

approximately

bp

length

from

the

mitochondrial

rRNA

gene

was

amplified

with

the

primer

-LRB-

CGY

AAG

GGA

AHG

CTG

-RRB-

which

has

a

single-base

modification

compared

with

Palumbi

et

al.

-LRB-

unpublished

manuscript

-RRB-

as

well

as

with

the

newly

designed

primer

-LRB-

CCG

GTC

TGA

ACT

CAG

ATC

ACG

-RRB-

Polymerase

chain

reaction

-LRB-

PCR

-RRB-

reactions

with

a

total

volume

of

μl

contained

μl

X

reaction

buffer

μl

dNTPs

-LRB-

mM

-RRB-

μl

of

each

primer

-LRB-

pmol/μl

-RRB-

μl

DNA-extract

μl

Teg

polymerase

-LRB-

U/μl

Prokaria

Reykjavik

Iceland

-RRB-

and

μl

deionized

water

Thermal

profile

began

at

°C

for

min

followed

by

cycles

of

°C

-LRB-

s

-RRB-

°C

-LRB-

s

-RRB-

°C

-LRB-

s

-RRB-

with

a

final

step

of

min

at

°C

PCR

products

were

purified

by

using

the

ExoSAP-IT

for

PCR

clean-up

-LRB-

GE

Healthcare

Uppsala

Sweden

-RRB-

The

newly

designed

sequencing

primer

-LRB-

TGC

ACC

ATT

AGG

ATG

TCC

TGA

TCC

-RRB-

was

used

to

sequence

one

strand

of

the

amplified

fragment

using

the

BigDye

Terminator

Cycle

Sequencing

Kit

-LRB-

ver

PE

Biosystems

Foster

City

USA

-RRB-

The

sequencing

reactions

were

run

in

an

ABI

Prism

automated

DNA

Analyzer

-LRB-

Applied

Biosystems

Foster

City

USA

-RRB-

according

to

the

manufacturer

's

instructions

Sequence

Analysis

and

Oligonucleotide

Probe

Design

In

the

framework

of

this

project

sequences

have

been

acquired

and

sequences

have

been

obtained

from

EMBL

sequence

database

and

other

projects

representing

approximately

species

of

fish

from

European

seas

Probes

were

designed

based

on

sequences

obtained

from

species

-LRB-

Table

-RRB-

A

multiple

alignment

of

these

sequences

was

performed

with

the

programme

Clustal

W

-LRB-

Thompson

et

al.

-RRB-

as

implemented

in

BioEdit

-LRB-

version

Hall

-RRB-

to

ensure

that

all

sequences

represent

a

homologous

fragment

of

the

rDNA

Before

probe

design

gaps

have

been

removed

from

each

sequence

A

computer

program

developed

by

the

bioinformatics

group

of

the

Centre

for

Applied

Gene

Sensor

Technology

-LRB-

CAG

-RRB-

and

the

Zentrum

für

Technomathematik

-LRB-

ZeTeM

-RRB-

both

at

University

of

Bremen

was

used

to

design

species-specific

oligonucleotide

probes

which

ideally

cover

all

sequences

of

one

species

and

do

not

match

any

other

species

-LRB-

Nölte

-RRB-

The

following

criteria

have

been

considered

-LRB-

-RRB-

length

of

to

bp

-LRB-

-RRB-

melting

temperature

-LRB-

Tm

-RRB-

of

to

°C

based

on

the

unified

model

-LRB-

SantaLucia

-RRB-

-LRB-

-RRB-

GC

content

of

to

-LRB-

-RRB-

secondary

structure

of

the

oligonucleotides

and

the

target

sequence

-LRB-

-RRB-

possible

dimer

formation

and

-LRB-

-RRB-

the

energy

content

of

a

bond

between

the

probe

and

the

target

sequence

Minimal

free

energy

-LRB-

mfe

-RRB-

structures

are

computed

by

using

RNAfold

-LRB-

Hofacker

et

al.

-RRB-

Probes

exhibiting

strong

secondary

structures

or

binding

to

a

region

of

the

target

with

such

a

strong

secondary

structure

were

not

used

If

more

than

one

probe

qualified

for

a

species

the

one

with

the

highest

binding

energy

between

probe

and

target

was

chosen

Already

during

the

design-phase

the

selected

oligonucleotide

probes

were

tested

in

silico

against

background

sequences

from

approximately

fish

species

Preparation

of

DNA

Microarrays

and

Hybridisation

Experiments

Aminosilane

-LRB-

-RRB-

coated

glass

slides

were

used

with

a

PDITC-linker

-LRB-

-RRB-

from

Asper

Biotech

-LRB-

Tartu

Estonia

-RRB-

Oligonucleotide

probes

-LRB-

Thermo

Hybaid

Ulm

Germany

-RRB-

with

a

were

spotted

in

mM

buffer

-LRB-

pH

-RRB-

at

a

concentration

of

μM

using

a

spotting

robot

based

on

a

modified

version

of

the

contactless

TopSpot

technology

The

spotted

volume

of

this

oligonucleotides

solution

was

pl

producing

a

spot

diameter

of

approximately

μm

Each

probe

was

spotted

in

four

replicates

per

block

An

array

contained

five

blocks

and

three

arrays

were

spotted

on

one

microarray

slide

-LRB-

Fig.

-RRB-

After

spotting

the

microarrays

were

incubated

for

h

in

a

wet

chamber

to

ensure

efficient

covalent

binding

of

the

oligonucleotides

Finally

the

microarrays

were

shrink-wrapped

under

a

nitrogen

atmosphere

and

were

storable

at

°C

for

up

to

months

Fig.

of

the

microarray

DNA

for

hybridisation

experiments

was

amplified

and

labelled

with

primers

The

primers

-LRB-

CGC

CTG

TTT

AAC

AAA

AAC

-RRB-

and

-LRB-

CCG

GTT

TGA

ACT

CAG

ATC

ACG

-RRB-

amplify

a

fragment

of

approximately

bp

length

from

the

mitochondrial

rRNA

gene

-LRB-

Palumbi

et

al.

unpublished

manuscript

-RRB-

PCR

reactions

with

a

volume

of

μl

contained

μl

X

reaction

buffer

μl

dNTPs

-LRB-

mM

-RRB-

μl

of

each

primer

-LRB-

μM

-RRB-

μl

DNA-extract

μl

Taq

polymerase

-LRB-

U/μl

-RRB-

μl

BSA

-LRB-

mg/ml

-RRB-

and

μl

deionized

water

The

PCR

thermal

profile

began

at

°C

for

min

followed

by

cycles

of

°C

-LRB-

s

-RRB-

°C

-LRB-

s

-RRB-

°C

-LRB-

s

-RRB-

followed

by

a

final

step

of

min

at

°C

The

PCR

amplified

DNA

was

purified

using

the

QIAquick

PCR

Purification

Kit

-LRB-

QIAGEN

Hilden

Germany

-RRB-

Hybridisation

experiments

were

performed

with

target

and

nontarget

fish

species

As

nontarget

species

fishes

were

chosen

that

are

closely

related

to

the

target

species

and

from

which

false-positive

signals

could

be

expected

according

to

the

in

silico

specificity

tests

performed

during

the

design

phase

of

the

probes

-LRB-

Table

-RRB-

Table

species

tested

in

hybridisation

experimentsSpeciesFamilyOrderDentex

dentexSparidaePerciformesDiplodus

vulgarisSparidaePerciformesGadus

morhuaGadidaeGadiformesMelanogrammus

aeglefinusGadidaeGadiformesMerlangius

merlangusGadidaeGadiformesMerluccius

merlucciusGadidaeGadiformesMicromesistius

poutassouGadidaeGadiformesMullus

surmuletusMullidaePerciformesPollachius

pollachiusGadidaeGadiformesPollachius

virensGadidaeGadiformesPsetta

maximaScophthalmidaePleuronectiformesSerranus

hepatusSerranidaePerciformesTrachurus

mediterraneusCarangidaePerciformesTrachuru

picturatusCarangidaePerciformesTaxonomy

according

to

FishBase

-LRB-

-RRB-

A

positive

control

at

a

concentration

of

nM

-LRB-

CGT

GTG

AGT

CGA

TGG

ATC

-RRB-

and

nM

of

the

purified

PCR

product

were

hybridized

to

the

microarray

in

a

volume

of

μl

using

GeneFrames

-LRB-

ABgene

House

Epsom

UK

-RRB-

which

were

applied

to

the

microarray

slides

according

to

the

manufacturer

's

instruction

-LRB-

Fig.

-RRB-

Hybridisation

was

conducted

at

°C

in

a

hybridisation

oven

After

h

hybridisation

time

the

GeneFrames

were

removed

and

the

microarrays

were

washed

minutes

each

with

SSC

-LRB-

sodium

chloride

trisodium

citrate

-RRB-

buffer

containing

SDS

-LRB-

sodium

dodecyl

sulphate

-RRB-

SSC

containing

SDS

and

SSC

Finally

the

microarrays

were

dried

in

a

centrifuge

at

rpm

for

minutes

Measurement

of

Fluorescence

Signals

and

Data

Analysis

Hybridisation

signals

were

measured

using

an

Axon

fluorescence

microarray

scanner

at

nm

-LRB-

-RRB-

as

well

as

at

nm

-LRB-

-RRB-

The

fluorescence

signal

analysis

was

conducted

with

the

software

GenePix

-LRB-

Axon

Union

City

USA

-RRB-

The

fluorescence

signals

of

each

probe

were

measured

and

the

arithmetic

mean

was

calculated

However

data

were

removed

from

the

analysis

if

the

spots

showed

artefacts

caused

during

the

spotting

process

-LRB-

e.g.

inhomogeneous

spots

documented

by

a

monitoring

camera

during

spotting

-RRB-

or

experimental

artefacts

-LRB-

e.g.

air

bubbles

-RRB-

Background

noise

was

corrected

by

subtracting

the

arithmetic

mean

of

the

negative

control

measurement

from

the

arithmetic

mean

of

the

spot

measurements

Negative

values

were

set

to

zero

Results

Probes

for

commercially

important

fish

species

have

been

designed

-LRB-

Table

-RRB-

Positions

of

the

oligonucleotide

probes

in

the

rDNA

fragment

used

for

probe

design

are

given

in

Fig.

Following

the

nomenclature

of

Ortí

et

al.

-LRB-

-RRB-

the

binding

sites

of

the

microarray

probes

for

Engraulis

encrasicolus

Sparus

aurata

and

Trigla

lyra

are

located

in

the

variable

region

j

whereas

the

probes

for

Boops

boops

Helicolenus

dactylopterus

Lophius

budegassa

Pagellus

acarne

Scomber

scombrus

Scophthalmus

rhombus

Serranus

cabrilla

and

Trachurus

trachurus

bind

to

the

variable

region

l.

Table

probes

for

the

identification

of

fish

species

from

European

seasSpecies

nameProbe

nameProbe

sequence

-LRB-

-RRB-

-LRB-

bp

-RRB-

Tm

-LRB-

°C

-RRB-

GC

-LRB-

-RRB-

Oligo

mfeDimer

mfeSpecificity

-LRB-

in

silico

-RRB-

Boops

boopsBooboo_315GCACCACACTCCTAAACCCAAGA2382

speciesEngraulis

encrasicolusEngenc_213CAAGTCCTAAATACCCGCAGCCT2382

speciesHelicolenus

dactylopterusHeldac_317ACCCCTCCTACAATTAAGAGCCG2381

speciesLophius

budegassaLopbud_312AACACCCTTCCTATCACCCAGAGCTAC2784

genusPagellus

acarnePagaca_317TACTACACTCCCACATCCGAGAGC2482

speciesScomber

scombrusScosco_321CAACTACTCCTACAGTCAAGAGCCACC2782

speciesScophthalmus

rhombusScorho_322CCCCTTAACTCCTCGAAGCAAGA2381

speciesSerranus

cabrillaSercab_313CCATTTTCCTACAACCCAGAGCGAC2582

speciesSparus

aurataSpaaur_201AGAACAGCTCACGTCAAACACCC2383

speciesTrachurus

trachurusTratra_333TTCCTCTCCTCCCACAAGCAAGA2383

genusTrigla

lyraTrilyr_232AAGACCGAACCAAATGAGCCCTG2383

familyThe

number

in

the

probe

name

indicates

the

binding

site

in

the

rDNA

sequenceOligo

mfe

minimal

free

energy

of

the

secondary

structure

of

the

oligonucleotide

Dimer

mfe

minimal

free

energy

of

the

dimer

of

two

identical

oligonucleotide

moleculesValues

for

mfe

are

given

in

kcal/molFig

-LRB-

-RRB-

of

representative

rDNA

sequences

from

the

target

species

with

binding

sites

-LRB-

light

grey

-RRB-

of

probes

-LRB-

probes

hybridise

to

the

reverse

complementary

target

strand

-RRB-

Double

stranded

-LRB-

dark

grey

-RRB-

and

single

stranded

regions

-LRB-

grey

-RRB-

of

the

secondary

structure

are

indicated

in

the

reference

sequence

of

Pygoplites

nattereri

-LRB-

Ortí

et

al.

Accession

number

-RRB-

All

single

target

hybridisations

of

the

rDNA

fragment

gave

true-positive

fluorescence

signals

for

the

corresponding

probe

-LRB-

Fig.

-RRB-

but

the

hybridisation

efficiency

is

very

different

The

signal

intensity

of

the

weakest

probe-target

pair

gave

approximately

arbitrary

units

and

the

strongest

almost

-LRB-

Table

-RRB-

Under

the

experimental

conditions

selected

single

target

hybridisations

with

B.

boops

E.

encrasicolus

H.

dactylopterus

L.

budegassa

S.

rhombus

S.

aurata

T.

trachurus

and

T.

lyra

did

not

show

any

false-positive

signals

Eight

probes

gave

under

these

conditions

very

weak

false-positive

signals

with

P.

acarne

S.

scombrus

and

S.

cabrilla

representing

approximately

of

all

possible

cross-hybridisation

reactions

The

values

of

these

false-positive

signals

were

several

orders

of

magnitude

lower

than

the

true

positive-signals

Figure

-LRB-

background

subtracted

from

absolute

signal

-RRB-

of

single

target

and

nontarget

hybridisations

White

bars

represent

true-positive

signals

false-positive

signals

are

shown

as

grey

bars

Numbers

at

the

basis

of

the

bars

indicate

the

amount

of

measured

spots

The

number

of

hybridisations

is

given

in

brackets

after

target

and

nontarget

names

Replication

and

absolute

signal

intensities

-LRB-

standard

deviation

-RRB-

of

hybridized

targets

to

the

corresponding

probe

are

given

in

Table

Table

hybridisationsHybridized

targetsNo

of

hybridisationsMeasurements

of

specific

probesMeasured

probes/absolute

no

of

probesMean

absolute

fluorescence

signal

in

arbitrary

unitsStandard

deviationBoops

Single

nontarget

hybridisations

showed

very

weak

false-positive

signals

representing

of

possible

cross-hybridisations

These

false-positive

signals

were

at

least

one

order

of

magnitude

weaker

than

the

true-positive

signal

Merlangius

merlangus

and

Merluccius

merluccius

did

not

gave

any

false-positive

signal

whereas

Dentex

dentex

Diplodus

vulgaris

Gadus

morhua

Melanogrammus

aeglefinus

Micromesistius

poutassou

Mullus

surmuletus

Pollachius

pollachius

Pollachius

virens

Psetta

maxima

Serranus

hepatus

Trachurus

mediterraneus

and

Trachurus

picturatus

showed

two

to

eight

cross-hybridisations

Considering

all

possible

cross-hybridisations

only

showed

usually

very

weak

false-positive

signals

Discussion

The

results

show

that

the

Fish

Chip

described

enables

the

identification

of

commercially

important

fish

species

from

European

seas

in

certain

experimental

limits

These

limits

are

primarily

given

by

the

fact

that

the

fluorescence

signal

intensities

of

true-positive

hybridisation

signals

were

heterogeneous

This

phenomenon

is

commonly

encountered

in

DNA

microarray

experiments

-LRB-

Peplies

et

al.

Warsen

et

al.

Korimbocus

et

al.

Rønning

et

al.

Tobler

et

al.

-RRB-

and

can

probably

be

overcome

only

by

an

extreme

methodical

effort

-LRB-

Shi

et

al.

-RRB-

The

problems

encountered

when

single-colour

microarray

experiments

need

to

be

quantified

are

severe

and

in

part

not

yet

solved

because

complex

parameters

are

influencing

the

results

These

are

the

sequence

dependent

hybridisation

efficiency

specifically

steric

hindrance

secondary

structures

-LRB-

Southern

et

al.

-RRB-

and

the

relative

position

of

the

fluorescent

label

at

the

target

-LRB-

Zhang

et

al.

-RRB-

Sometimes

the

duplex

formation

can

be

favoured

by

using

spacers

which

obviously

give

the

captures

a

greater

degree

of

freedom

from

their

neighbouring

molecules

and

from

the

surface

-LRB-

Southern

et

al.

-RRB-

leading

to

an

enhancement

of

signal

intensity

with

increasing

spacer

length

-LRB-

Peplies

et

al.

-RRB-

An

important

criterion

of

probe

design

was

the

base

composition

-LRB-

Southern

et

al.

-RRB-

and

therefore

all

but

one

capture

oligonucleotides

were

designed

to

have

a

GC

content

of

and

for

the

exception

-LRB-

Table

-RRB-

The

different

hybridisation

efficiencies

of

the

captures

and

the

varying

sensitivity

for

the

different

fishes

is

still

a

severe

disadvantage

because

it

can

hamper

the

estimation

of

a

small

amount

of

fishes

of

one

species

in

the

presence

of

a

larger

number

of

individuals

of

another

species

in

a

mixed

sample

These

limits

are

presently

under

investigation

with

fish

eggs

and

other

biological

material

Because

all

oligonucleotide

probes

bind

to

the

variable

regions

j

and

l

of

the

rRNA

gene

which

represent

large

single-stranded

loops

the

secondary

structure

is

unlikely

to

be

a

factor

contributing

to

the

partially

low

sensitivity

Also

the

POL

effect

a

phenomenon

decreasing

the

fluorescence

signal

with

increasing

distance

between

the

probe

binding

site

and

the

label

on

the

target

-LRB-

Zhang

et

al.

-RRB-

seems

not

to

be

important

in

this

case

Most

of

the

probes

used

in

this

study

hybridise

to

the

variable

region

l

of

the

rDNA

and

their

distance

from

the

fluorescence

label

is

more

or

less

identical

Although

cross-hybridisations

occur

true-positive

signals

could

clearly

be

differentiated

from

false-positive

signals

because

of

their

generally

higher

signal

Most

false-positive

signals

occurred

when

the

rDNA

fragment

of

nontarget

species

was

hybridised

on

the

microarray

Testing

of

nontarget

species

is

seemingly

important

in

studies

using

DNA

microarrays

for

the

identification

of

organisms

because

even

if

a

comprehensive

sequence

background

is

utilised

for

probe

design

and

even

if

extensive

in

silico

testing

has

been

performed

the

specificity

of

oligonucleotide

probes

has

to

be

evaluated

with

closely

related

species

and

specifically

those

that

show

cross-hybridisations

in

silico

This

study

shows

that

the

rRNA

gene

of

fishes

is

suitable

to

design

oligonucleotide

probes

that

are

able

to

differentiate

eleven

fish

species

from

European

seas

by

single

target

hybridisation

on

a

microarray

Such

a

Fish

Chip

can

hopefully

be

applied

in

marine

environmental

and

fisheries

research

as

well

as

in

fisheries

and

food

control

if

the

uneven

hybridisation

signal

intensities

of

the

different

probe-target

pairs

can

be

improved

or

compensated

The

correct

identification

of

fish

eggs

and

larvae

is

crucial

for

fish

stock

assessment

based

on

ichthyoplankton

surveys

Genetic

identification

has

shown

that

the

majority

of

eggs

in

the

Irish

Sea

wrongly

believed

to

be

from

cod

were

actually

from

whiting

leading

to

an

overestimation

of

cod

stocks

-LRB-

Fox

et

al.

-RRB-

A

study

on

food

fish

in

the

United

States

revealed

that

three-quarters

of

fish

sold

as

red

snapper

were

mislabelled

and

belonged

to

other

species

-LRB-

Marko

et

al.

-RRB-

a

situation

that

needs

better

analytical

tools

to

be

changed

The

European

Union

-LRB-

EU

-RRB-

also

has

strict

regulations

for

seafood

labelling

which

must

include

for

example

the

species

name

-LRB-

EU

Council

Regulation

No

EU

Commission

Regulation

No

-RRB-

Approximately

species

of

fish

are

sold

in

the

German

market

alone

making

a

reliable

identification

method

urgently

necessary

to

protect

the

customer

DNA

microarrays

might

have

the

potential

to

fulfill

these

requirements

Recent

efforts

in

compiling

sequences

of

fishes

such

as

the

European

projects

FishTrace

-RRB-

and

Fish

Chips

-LRB-

Kochzius

et

al.

-RRB-

as

well

as

the

international

Fish

Barcode

of

Life

Initiative

Ward

et

al.

-RRB-

will

provide

the

necessary

sequence

background

for

the

design

of

species

specific

oligonucleotide

probes

for

the

development

of

DNA

microarrays

for

the

identification

of

fishes

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

dna chips
species identification
capture oligonucleotide
pisces