Monitoring

of

platinum

surface

contamination

in

seven

Dutch

hospital

pharmacies

using

inductively

coupled

plasma

mass

spectrometry

Objective

To

develop

validate

and

apply

a

method

for

the

determination

of

platinum

contamination

originating

from

cisplatinum

oxaliplatinum

and

carboplatinum

Introduction

Cytotoxic

drugs

are

widely

used

for

the

treatment

of

cancer

Occupational

exposure

to

these

drugs

has

been

recognised

as

a

potential

health

hazard

since

-LRB-

Donner

Falck

et

al.

-RRB-

Because

cytotoxic

drugs

can

affect

the

DNA

RNA

or

protein

synthesis

many

of

these

drugs

are

classified

as

being

carcinogenic

mutagenic

or

teratogenic

to

humans

-LRB-

International

Agency

for

Research

on

Cancer

-LRB-

IARC

-RRB-

-RRB-

Skin

contact

with

cytotoxic

drugs

due

to

contamination

of

the

work

area

or

contamination

of

packaging

material

seems

to

play

an

important

role

in

the

uptake

of

these

drugs

by

hospital

personnel

-LRB-

Fransman

et

al.

Sessink

et

al.

-RRB-

Therefore

strict

health

and

safety

rules

have

been

established

and

applied

for

the

handling

of

these

agents

Evidently

the

potential

health

risks

for

persons

manipulating

cytotoxic

drugs

such

as

pharmacists

pharmacy

technicians

nurses

and

cleaners

however

still

are

a

concern

This

concern

is

consolidated

by

a

number

of

recent

publications

demonstrating

workplace

contamination

-LRB-

Crauste-Manciet

et

al.

Hedmer

et

al.

Leboucher

et

al.

Mason

et

al.

Schmaus

et

al.

Zeedijk

et

al.

Ziegler

et

al.

-RRB-

and

contamination

of

packaging

of

cytotoxic

drugs

-LRB-

Connor

et

al.

Hedmer

et

al.

Mason

et

al.

Nygren

et

al.

-RRB-

Moreover

detection

of

cytotoxic

agents

in

urine

-LRB-

Ensslin

et

al.

b

Minoia

et

al.

Pethran

et

al.

Schreiber

et

al.

Sessink

et

al.

Turci

et

al.

-RRB-

and

blood

-LRB-

Nygren

and

Lundgren

-RRB-

of

personnel

who

were

involved

in

preparation

or

administration

has

been

reported

with

increasing

frequency

The

relationship

between

prolonged

exposure

to

small

quantities

of

cytotoxic

drugs

and

harmful

effects

is

difficult

to

establish

Based

on

current

scientific

knowledge

it

is

impossible

to

set

a

level

of

exposure

that

beyond

doubt

will

not

cause

adverse

effects

Because

no

regulations

on

the

maximal

acceptable

amount

of

contamination

for

these

drugs

have

been

set

so

far

hospitals

should

aim

for

the

lowest

contamination

as

is

reasonably

achievable

Monitoring

of

contamination

therefore

is

essential

This

can

aid

in

the

identification

of

the

main

exposure

routes

and

in

assessing

the

effectiveness

of

cleaning

and

working

procedures

Evaluation

of

environmental

contamination

will

moreover

lead

to

an

increase

of

the

consciousness

among

personnel

concerning

the

handling

of

the

chemotherapeutic

agents

This

can

lead

to

an

improvement

of

and

the

compliance

to

working

and

cleaning

procedures

Wipe

sampling

is

a

common

method

to

monitor

surfaces

for

the

presence

of

cytotoxic

drugs

Hence

sensitive

and

validated

methods

are

indispensable

to

be

able

to

detect

the

relatively

low

quantities

of

drug

present

on

surfaces

Platinum

coordination

complexes

such

as

cisplatinum

oxaliplatinum

and

carboplatinum

play

a

major

role

in

the

treatment

of

a

variety

of

tumours

As

a

result

large

amounts

of

these

agents

are

processed

in

hospital

pharmacies

Several

wipe

sample

methods

for

platinum

containing

drugs

have

been

used

in

earlier

studies

and

platinum

was

detected

as

a

surface

contaminant

in

many

of

the

workplaces

-LRB-

Leboucher

et

al.

Mason

et

al.

Schmaus

et

al.

Ziegler

et

al.

-RRB-

or

drug

vials

-LRB-

Connor

et

al.

Nygren

et

al.

-RRB-

investigated

A

description

of

the

validation

of

the

analytical

methods

however

has

been

scarce

Validation

results

were

mentioned

briefly

for

the

method

of

Ziegler

et

al.

-LRB-

-RRB-

using

electro

thermal

vaporisation

coupled

to

inductively

coupled

plasma

mass

spectrometry

-LRB-

ICP-MS

-RRB-

Raghavan

et

al.

-LRB-

-RRB-

described

the

validation

of

a

high-performance

liquid

chromatography

method

for

the

determination

of

cisplatinum

in

cleaning

validation

samples

The

limit

of

quantification

of

this

method

was

ng

l

which

is

high

compared

to

the

limits

achievable

with

for

example

ICP-MS

or

voltammetry

Schmaus

et

al.

-LRB-

-RRB-

reported

the

validation

of

a

voltammetric

method

with

a

limit

of

quantification

of

pg

of

platinum

per

sample

In

the

present

study

we

describe

the

development

and

validation

of

an

ICP-MS

method

for

the

evaluation

of

surface

contamination

by

platinum

originating

from

cisplatinum

oxaliplatinum

and

carboplatinum

ICP-MS

assures

an

ultra

high

sensitivity

and

specificity

and

requires

relatively

simple

sample

pre-treatment

procedures

The

validated

method

has

been

applied

to

measure

surface

contamination

in

seven

Dutch

hospital

pharmacies

Experimental

Chemicals

Cisplatinum

and

carboplatinum

reference

standards

were

purchased

from

Calbiochem

-LRB-

San

Diego

CA

USA

-RRB-

Oxaliplatinum

was

obtained

from

Sigma-Aldrich

-LRB-

St

Louis

MO

USA

-RRB-

Chloroplatinic

acid

containing

mg

l

platinum

in

HCl

used

for

preparation

of

calibration

solutions

was

obtained

from

Inorganic

Ventures/IV

Labs

-LRB-

Lakewood

NJ

USA

-RRB-

Iridium

chloride

containing

mg

l

iridium

in

HCl

used

for

internal

standardisation

was

also

purchased

from

Inorganic

Ventures/IV

Labs

Nitric

acid

-LRB-

-RRB-

and

hydrochloric

acid

-LRB-

HCl

-RRB-

Ultrex

II

ultrapure

reagents

were

obtained

from

Mallinckrodt

Baker

-LRB-

Philipsburg

NJ

USA

-RRB-

Water

used

for

the

ICP-MS

analysis

was

sterile

water

for

irrigation

-LRB-

Aqua

B.

Braun

Medical

Melsungen

Germany

-RRB-

Ethanol

was

purchased

from

Fresenius

Kabi

-LRB-

Den

Bosch

The

Netherlands

-RRB-

A

multi-element

solution

containing

mg

l

of

Ba

Be

Ce

Co

In

Mg

Pb

Th

Tl

-LRB-

VAR-TS-MS

-RRB-

was

purchased

from

Inorganic

Ventures/IV

Labs

Hoek

Loos

-LRB-

Schiedam

The

Netherlands

-RRB-

provided

argon

gas

-LRB-

-RRB-

with

purity

Instrumentation

Analyses

were

performed

on

an

ICP-quadrupole-MS

-LRB-

Varian

-RRB-

equipped

with

a

reflecting

ion

mirror

-LRB-

Varian

Mulgrave

Victoria

Australia

-RRB-

The

sample

introduction

system

consisted

of

a

Micromist

glass

low

flow

nebuliser

-LRB-

sample

uptake

ml

min

-RRB-

a

peltier-cooled

-LRB-

°C

-RRB-

double

pass

glass

spray

chamber

and

a

quartz

torch

The

spray

chamber

was

cooled

to

reduce

the

vapour

loading

on

the

plasma

increasing

the

available

energy

for

atomisation

and

ionisation

of

the

elements

of

interest

Sample

transport

from

the

autosampler

-LRB-

Varian

-RRB-

to

the

nebuliser

was

performed

using

a

peristaltic

pump

-LRB-

Watson-Marlow

Alitea

Stockholm

Sweden

-RRB-

The

instrument

was

cooled

by

using

a

Kühlmobil

VD

-LRB-

Van

der

Heijden

Dörentrup

Germany

-RRB-

Data

were

acquired

and

processed

using

the

ICP-MS

Expert

Software

version

-LRB-

Varian

-RRB-

Further

data

handling

was

performed

using

Excel

-LRB-

Microsoft

Redmond

WA

USA

-RRB-

All

measurements

were

carried

out

in

a

dedicated

temperature-controlled

positively

pressurised

environment

in

order

to

maintain

optimum

instrument

performance

and

minimise

exogenous

contamination

All

solutions

were

prepared

using

pipettes

-LRB-

Falcon

Becton

Dickinson

Labware

Franklin

Lakes

NJ

USA

-RRB-

and

polypropylene

tubes

ml

-LRB-

Plastiques-Gosselin

Hazebrouck

Cedex

France

-RRB-

and

ml

-LRB-

Sarstedt

AG&C

o

Nümbrecht

Germany

-RRB-

Filters

-LRB-

Minisart

-RRB-

used

for

filtration

of

wipe

samples

were

obtained

from

Sartorius

-LRB-

Hannover

Germany

-RRB-

Prior

to

method

development

tubes

were

checked

thoroughly

for

platinum

iridium

and

hafnium

contamination

and

appeared

to

be

suitable

for

platinum

analyses

Determination

of

platinum

by

ICP-MS

To

optimise

the

ICP-MS

signal

for

the

high

masses

and

to

reduce

the

formation

of

oxides

a

solution

containing

ng

l

of

Th

In

Ce

Ba

and

Pt

was

used

Typically

this

ng

l

solution

gave

readings

of

c

s

c

s

and

Pt

c

s

The

production

of

CeO

was

less

than

of

the

total

Ce

counts

The

formation

of

doubly

charged

Ba

was

less

than

Performance

was

checked

daily

The

platinum

isotope

used

for

calculation

of

platinum

concentrations

was

Pt

Iridium

was

used

as

internal

standard

Detection

of

platinum

can

be

subject

to

the

interference

of

hafmium

oxides

-LRB-

Lustig

et

al.

-RRB-

Therefore

hafmium

signals

were

monitored

for

all

samples

The

detection

mode

for

all

isotopes

was

based

on

peak

jumping

with

peak

dwell

times

of

ms

scans

per

replicate

and

three

replicates

per

sample

Quantitation

was

based

on

the

mean

concentration

of

three

replicates

analysed

against

a

calibration

curve

using

weighted

linear

regression

analysis

Assay

development

The

most

suitable

wipe

material

desorption

solvent

and

wipe

solvent

were

selected

using

one

surface

sampling

and

desorption

procedure

This

will

be

described

below

Recovery

data

were

assessed

for

the

three

most

commonly

used

platinum

agents

cisplatinum

oxaliplatinum

and

carboplatinum

The

different

molecular

structures

and

as

a

result

the

variable

physical

characteristics

might

possibly

lead

to

a

variation

in

absorption

and

desorption

characteristics

Therefore

we

decided

to

evaluate

all

three

compounds

instead

of

choosing

one

reference

compound

Surface

sampling

and

desorption

procedure

Each

wipe

tissue

was

moistened

with

μl

wipe

solvent

In

general

sampling

was

performed

by

wiping

a

defined

surface

area

of

cm

cm

However

for

surfaces

for

which

it

was

not

possible

to

take

a

cm

cm

sample

the

complete

top

of

the

device

was

sampled

and

the

area

was

estimated

All

wipe

samples

were

collected

with

a

uniform

sampling

procedure

by

wiping

in

three

different

directions

-LRB-

vertical

horizontal

and

diagonal

-RRB-

Wipe

samples

were

stored

in

ml

disposable

polypropylene

flasks

-LRB-

Falcon

Becton

Dickinson

Labware

Franklin

Lakes

NJ

USA

-RRB-

at

°C

until

further

processing

Prior

to

analysis

ml

of

desorption

solvent

was

added

to

the

sample

and

flasks

were

kept

in

an

ultrasonic

bath

for

min

Then

samples

were

filtered

to

remove

particles

which

could

possibly

obstruct

the

ICP-MS

nebuliser

or

could

interfere

with

the

analysis

Two

millilitres

of

sample

were

after

addition

of

iridium

as

internal

standard

introduced

directly

into

the

ICP-MS

Samples

of

locations

which

were

expected

to

be

highly

contaminated

were

diluted

prior

to

analysis

to

prevent

contamination

of

the

sample

introduction

system

of

the

ICP-MS

Wipe

material

A

variety

of

wipe

tissues

are

available

for

collecting

samples

of

surface

contaminants

These

vary

in

type

of

material

surface

area

and

content

of

platinum

contaminants

Three

types

of

wipes

were

evaluated

for

this

study

Kimtech

Science

precision

wipes

-LRB-

Kimberley-Clark

Professional

Irving

TX

USA

-RRB-

Whatman

glass

fibre

filters

-LRB-

Schleicher

Schuell

Microscience

GmbH

Dassel

Germany

-RRB-

and

Klinion

non-woven

gauzes

-LRB-

Medeco

Oud-Beijerland

The

Netherlands

-RRB-

The

tissues

were

checked

for

platinum

contamination

and

for

their

ability

to

release

platinum

from

stainless

steel

surfaces

Desorption

solvent

One

percent

-LRB-

v/v

-RRB-

-LRB-

v/v

-RRB-

and

HCl

-LRB-

v/v

-RRB-

were

evaluated

as

desorption

solutions

Kimtech

Science

precision

wipes

were

spiked

with

cisplatinum

oxaliplatinum

and

carboplatinum

and

the

platinum

recovery

was

determined

after

desorption

with

ml

of

each

solvent

Wipe

solvent

Initially

water

HCl

and

ethanol

were

selected

as

wiping

solutions

To

investigate

the

capability

of

these

solutions

to

effectively

wipe

surfaces

stainless

steel

surfaces

were

spiked

with

cisplatinum

oxaliplatinum

and

carboplatinum

These

surfaces

were

subsequently

wiped

and

samples

were

desorbed

using

a

HCl

solution

Validation

procedures

Linearity

For

calibration

the

chloroplatinic

acid

reference

solution

containing

mg

l

platinum

was

diluted

with

HCl

to

obtain

working

solutions

with

concentrations

ranging

from

to

ng

l

platinum

Working

solutions

were

diluted

with

HCl

to

obtain

calibration

standards

ranging

from

to

ng

l

platinum

Before

analysis

μl

of

iridium

internal

standard

solution

was

added

to

ml

of

each

calibration

standard

-LRB-

final

internal

standard

concentration

ng

l

-RRB-

The

seven

non-zero

calibration

standards

were

processed

and

analysed

in

singular

in

three

separate

analytical

runs

The

calibrations

were

back-calculated

from

the

responses

Deviations

from

the

nominal

concentration

were

evaluated

Recovery

and

precision

Quality

control

-LRB-

QC

-RRB-

samples

were

prepared

to

obtain

information

on

the

recovery

and

precision

of

the

desorption

method

and

platinum

analysis

These

samples

were

analysed

in

the

validation

runs

and

subsequently

also

during

the

analysis

of

the

wipe

samples

of

each

hospital

pharmacy

Therefore

stock

solutions

of

the

platinum

agents

in

water

each

containing

a

concentration

of

drug

equivalent

to

mg

l

platinum

were

prepared

These

stock

solutions

were

further

diluted

to

obtain

spiking

solutions

with

concentrations

ranging

from

to

ng

l

Tissues

were

spiked

with

these

solutions

serving

as

QC

samples

at

the

following

concentration

levels

and

ng

platinum

on

the

tissues

corresponding

to

and

ng

l

platinum

in

the

final

solution

These

tissues

were

processed

as

described

earlier

Three

replicates

of

each

sample

were

analysed

in

three

analytical

runs

Recovery

was

expressed

as

a

percentage

of

the

nominal

concentration

Within-run

and

between-run

precisions

were

calculated

by

analysis

of

variances

-LRB-

ANOVA

-RRB-

for

each

test

concentration

using

the

analytical

run

as

the

grouping

variable

Two

of

the

most

important

surfaces

stainless

steel

and

linoleum

were

used

to

obtain

information

on

the

recovery

and

within-run

and

between-run

precisions

of

the

complete

sampling

procedure

including

both

wiping

and

desorption

Therefore

ng

of

cisplatinum

oxaliplatinum

or

carboplatinum

was

pipetted

in

triplicate

on

a

stainless

steel

and

linoleum

surface

After

drying

overnight

surfaces

were

wiped

following

the

previously

described

procedure

and

analysed

using

ICP-MS

Three

replicates

of

each

sample

were

analysed

in

three

analytical

runs

for

both

surfaces

Recovery

was

expressed

as

a

percentage

of

the

nominal

concentration

Within-run

and

between-run

precision

were

calculated

by

ANOVA

for

each

test

concentration

using

the

analytical

run

as

the

grouping

variable

Sensitivity

The

lower

limit

of

quantitation

-LRB-

LLOQ

-RRB-

was

defined

as

the

concentration

at

which

the

analyte

response

was

at

least

five

times

the

response

of

a

blank

wipe

sample

Besides

the

LLOQ

when

spiked

on

blank

tissues

had

to

be

determined

with

a

precision

less

than

and

the

mean

value

should

not

deviate

more

than

of

the

actual

value

Stability

Stability

of

cisplatinum

oxaliplatinum

and

carboplatinum

spiked

to

tissues

at

two

concentration

levels

was

evaluated

at

ambient

temperatures

for

week

and

under

storage

conditions

-LRB-

°C

-RRB-

for

up

to

weeks

From

each

storage

condition

two

wipe

samples

were

analysed

Samples

were

considered

stable

when

of

the

initial

concentration

was

recovered

Platinum

determination

in

pharmacy

facilities

where

no

cytotoxic

agents

are

processed

Platinum

is

an

element

that

not

only

appears

in

the

environment

due

to

contamination

with

platinum

containing

cytotoxic

drugs

but

also

due

to

pollution

by

car

exhaust

catalysts

As

a

consequence

road

dust

also

contains

platinum

-LRB-

Barefoot

-RRB-

Even

though

in

the

pharmacy

facilities

precautions

-LRB-

use

of

slippers/clogs

-RRB-

are

taken

to

reduce

the

chance

of

contamination

of

the

facility

road

dust

contamination

might

occur

Because

ICP-MS

does

not

differentiate

between

the

sources

of

elemental

platinum

this

should

be

taken

into

account

when

considering

this

technique

for

evaluation

of

environmental

contamination

by

cytotoxic

platinum

agents

Therefore

two

additional

locations

were

included

in

this

study

to

set

a

threshold

below

which

it

was

not

possible

to

address

the

source

of

the

contamination

The

first

location

was

the

laminar-airflow

-LRB-

LAF

-RRB-

hood

in

a

clean

room

in

which

no

platinum

contamination

was

expected

The

second

location

was

a

preparation

unit

with

LAF

hood

of

a

public

pharmacy

Monitoring

of

surface

contamination

in

seven

Dutch

hospital

pharmacies

The

wipe

samples

were

taken

in

seven

hospital

pharmacies

in

The

Netherlands

Characteristics

of

the

facilities

are

shown

in

Table

The

facilities

were

selected

to

provide

a

representation

of

the

diversity

in

hospital

pharmacies

in

The

Netherlands

in

terms

of

size

and

amount

of

platinum

compounds

handled

The

facilities

of

each

hospital

consisted

of

a

preparation

room

with

at

least

one

LAF

hood

and

a

room

for

storage

and

checking

of

the

prepared

drugs

and

administration

purposes

In

each

facility

samples

were

taken

at

locations

that

were

prone

to

contamination

For

good

comparison

of

the

results

standard

locations

-LRB-

Fig.

-RRB-

were

selected

-LRB-

-RRB-

the

middle

of

the

bench-top

of

the

LAF

hood

-LRB-

-RRB-

front

edge

of

LAF

hood

-LRB-

-RRB-

floor

in

front

of

LAF

hood

-LRB-

-RRB-

handle

of

service

hatch

-LRB-

-RRB-

door

handle

-LRB-

-RRB-

waste

bin

top

-LRB-

-RRB-

bench-top

on

which

materials

are

placed

in

storage/checking

room

-LRB-

-RRB-

floor

in

front

of

-LRB-

-RRB-

-LRB-

-RRB-

mouse

computer

-LRB-

-RRB-

handle

telephone

-LRB-

-RRB-

storage

shelve

of

cisplatinum

-LRB-

-RRB-

storage

shelve

oxaliplatinum

-LRB-

-RRB-

storage

shelve

carboplatinum

-LRB-

-RRB-

transport

box

-LRB-

-RRB-

handle

refrigerator

Locations

and

were

sampled

in

duplicate

to

get

an

impression

of

the

overall

contamination

of

these

locations

A

new

pair

of

gloves

was

used

for

each

wipe

sample

For

each

facility

three

blank

samples

were

prepared

by

moistening

tissue

with

μl

water

All

samples

were

stored

and

processed

as

described

earlier

The

storage

time

from

sampling

until

work-up

procedure

was

less

than

weeks

Table

of

platinum

agents

processed

and

years

that

the

facilities

are

in

use

in

-LRB-

in

g

-RRB-

Oxaliplatinum

use

in

-LRB-

in

g

-RRB-

Carboplatinum

use

in

-LRB-

in

g

-RRB-

Total

amount

of

platinum

processed

in

-LRB-

in

g

-RRB-

Number

of

years

in

Wipe

sampling

frequencyOnce

per

first

timeSporadic

last

in

per

yearOnce

per

yearOnce

per

year

Twice

per

yearFig

locations

in

pharmacy

facilities

Wipe

sampling

was

announced

in

each

facility

in

advance

and

was

performed

after

the

daily

cleaning

procedure

of

the

LAF

hoods

but

before

the

daily

cleaning

procedure

of

the

rest

of

the

facility

Wipe

sampling

in

all

the

facilities

was

performed

by

the

same

person

Results

Assay

development

Wipe

material

As

a

result

of

high

platinum

backgrounds

-LRB-

pg

platinum

per

filter

depending

on

the

batch

analysed

-RRB-

Whatman

glass

fibre

filters

were

found

to

be

not

suitable

for

platinum

wipe

sampling

Kimtech

Science

precision

wipes

and

Klinion

non-woven

gauzes

did

not

show

platinum

contamination

However

Kimtech

Science

precision

wipes

showed

better

recoveries

of

platinum

compared

to

Klinion

non-woven

gauzes

and

consequently

Kimtech

Science

precision

wipes

appeared

to

be

the

best

choice

Desorption

solvent

The

most

effective

desorption

of

platinum

from

the

wipe

materials

was

achieved

by

HCl

-LRB-

-RRB-

As

a

result

HCl

was

selected

as

the

desorption

solution

of

choice

Wipe

solvent

Recoveries

were

inadequate

for

ethanol

-LRB-

for

all

three

compounds

-RRB-

and

acceptable

for

water

-LRB-

-RRB-

and

HCl

-LRB-

-RRB-

Because

HCl

appeared

to

be

corrosive

for

some

types

of

stainless

steel

water

was

selected

as

wipe

solution

Validation

procedures

Linearity

The

calibration

curve

was

best

described

by

linear

regression

using

-LRB-

sd

of

triplicate

sample

reading

-RRB-

as

weight-factor

to

avoid

bias

in

favour

of

samples

with

high

standard

deviations

Deviations

from

the

nominal

concentration

were

between

and

for

all

concentration

levels

Relative

standard

deviations

for

the

calibration

samples

were

up

to

Correlation

coefficients

were

higher

than

Recovery

and

precision

Within-run

and

between-run

precision

data

for

spiked

tissues

which

served

as

QCs

are

summarised

in

Table

Precision

data

showed

that

for

all

QC

concentration

levels

the

reproducibility

of

the

desorption

procedure

and

platinum

analysis

was

excellent

Recoveries

for

cisplatinum

oxaliplatinum

and

carboplatinum

were

between

and

for

all

concentration

levels

These

results

indicated

sufficient

recovery

Table

and

between-run

precision

data

for

quality

control

samplesAmount

of

platinum

spiked

to

tissue

-LRB-

in

ng

-RRB-

Final

platinum

concentration

-LRB-

in

ng

l

-RRB-

.10010.01.357.441.751.272.862.501.001001.077.791.63

aNo

statistically

significant

additional

value

was

observed

as

a

result

of

performing

the

assay

in

different

runs

-LRB-

mean

square

within

runs

is

greater

than

mean

square

between

runs

-RRB-

For

recovery

and

within-run

and

between-run

precision

data

from

the

spiked

stainless

steel

and

linoleum

surfaces

see

respectively

Tables

and

Precision

data

showed

that

the

reproducibility

of

the

method

including

the

wiping

procedure

was

good

Recoveries

from

the

spiked

stainless

steel

surface

were

for

cisplatinum

for

oxaliplatinum

and

for

carboplatinum

-LRB-

Table

-RRB-

Recoveries

for

the

linoleum

surface

were

respectively

and

-LRB-

Table

-RRB-

Table

of

ng

platinum

from

a

stainless

steel

surfaceCisplatinumOxaliplatinumCarboplatinumMean

recovery

-LRB-

-RRB-

precision

-LRB-

in

-RRB-

precision

-LRB-

in

-RRB-

aaNumber

of

of

samples

per

statistically

significant

additional

value

was

observed

as

a

result

of

performing

the

assay

in

different

runs

-LRB-

mean

square

within

runs

is

greater

than

mean

square

between

runs

-RRB-

Table

of

ng

platinum

from

a

linoneum

recovery

-LRB-

-RRB-

precision

-LRB-

in

-RRB-

precision

-LRB-

in

-RRB-

Number

of

of

samples

per

Sensitivity

The

LLOQ

of

the

assay

was

set

at

a

platinum

concentration

of

ng

l

in

HCl

corresponding

to

pg

per

wipe

sample

or

pg

cm

taking

into

account

a

surface

of

cm

cm

Signal

to

noise

ratios

at

the

LLOQ

level

exceeded

during

all

the

experiments

which

was

in

accordance

with

the

requirement

The

acceptance

criteria

that

the

LLOQ

was

determined

with

a

precision

less

than

and

that

the

mean

value

should

deviate

no

more

than

from

the

actual

value

were

met

for

all

three

compounds

-LRB-

Table

-RRB-

Stability

Stability

has

now

been

established

up

to

weeks

but

further

testing

is

still

ongoing

Sample

storage

at

room

temperature

for

week

was

not

possible

Tissues

which

were

spiked

with

cisplatinum

showed

a

decrease

in

platinum

levels

of

after

week

Oxaliplatinum

and

carboplatinum

spiked

tissues

did

not

reduce

under

these

conditions

Sample

storage

at

°C

was

possible

for

at

least

weeks

Platinum

concentrations

of

cisplatinum

spiked

tissues

were

decreasing

more

obvious

with

time

than

oxaliplatinum

and

carboplatinum

spiked

tissues

However

no

decrease

of

more

than

of

the

initial

concentration

was

observed

after

weeks

at

°C

Platinum

determination

in

pharmacy

facilities

where

no

cytotoxic

agents

are

processed

No

platinum

was

detected

in

wipe

samples

from

the

LAF

hood

of

the

public

pharmacy

Platinum

levels

of

the

LAF

hood

in

the

clean

room

and

the

floor

in

the

public

pharmacy

ranged

between

and

ng

l

-LRB-

or

pg

cm

-RRB-

Therefore

it

was

recommended

to

set

a

threshold

of

ng

l

platinum

-LRB-

pg

cm

when

wiping

a

surface

of

-RRB-

below

which

it

was

not

possible

to

address

the

source

of

the

contamination

All

surfaces

in

the

preparation

units

with

platinum

levels

above

this

threshold

were

considered

as

being

contaminated

by

platinum

containing

drugs

Monitoring

surface

contamination

in

seven

Dutch

hospital

pharmacies

In

February

wipe

samples

were

collected

from

seven

Dutch

hospital

pharmacies

with

centralised

units

dedicated

to

the

preparation

of

intravenous

mixtures

of

cytotoxic

drugs

The

amount

of

platinum

which

was

processed

in

these

facilities

ranged

from

to

g

per

year

-LRB-

Table

-RRB-

Surface

contamination

of

all

sample

locations

is

depicted

in

Table

in

pg

cm

It

is

important

to

consider

that

recoveries

of

the

samples

as

assessed

in

the

validation

study

deviate

from

dependent

on

the

type

of

surface

sampled

and

on

the

type

of

compounds

present

on

the

surface

Therefore

results

depicted

in

Table

represent

of

the

actual

contamination

present

on

the

surface

Table

contamination

in

seven

Dutch

hospital

pharmaciesS.no.Sampled

surfacePlatinum

contamination

-LRB-

in

pg

cm

-RRB-

of

bench

LAF

Duplicate

of

Front

edge

of

LAF

hood

Duplicate

of

Floor

in

front

of

LAF

Duplicate

of

Handle

of

service

hatch

Door

handle

Waste

bin

top

on

which

materials

are

Floor

in

front

of

Mouse

Handle

telephone

Storage

shelve

Storage

shelve

Storage

shelve

Transport

aRecovered

platinum

concentrations

were

below

the

thresholdbDevice

was

not

present

of

available

for

wipe

samplingcHafnium

oxide

might

have

accounted

for

up

to

of

the

platinum

content

Platinum

was

detected

in

of

the

wipe

samples

and

of

the

samples

contained

levels

above

the

threshold

set

Six

of

the

samples

showed

raised

hafmium

signals

which

considering

a

maximum

oxide

formation

of

might

have

accounted

for

up

to

of

the

platinum

signals

of

these

samples

of

blank

samples

prepared

for

each

facility

by

moistening

tissues

with

wipe

solvent

contained

levels

of

platinum

exceeding

of

the

LLOQ

standard

The

variation

in

the

level

of

contamination

between

pharmacies

was

high

Pharmacies

of

site

and

showed

overall

low

platinum

contaminations

For

these

sites

respectively

and

did

not

contain

platinum

levels

above

the

threshold

set

Platinum

levels

detected

at

pharmacy

were

relatively

low

as

well

although

the

wipes

taken

from

the

floor

were

high

at

this

site

These

high

values

were

most

probably

the

result

of

a

calamity

in

with

a

cisplatinum

infusion

mixture

which

was

spilled

on

the

floor

Most

locations

wiped

at

the

hospital

pharmacies

of

site

and

showed

high

contaminations

Only

one

sample

from

these

sites

did

not

contain

any

detectable

platinum

The

high

contamination

of

site

seemed

to

run

counter

to

the

quantities

of

drugs

handled

because

in

this

pharmacy

relatively

low

amounts

of

platinum

were

processed

This

site

however

was

occasionally

used

for

preparation

of

larger

amounts

of

cytotoxic

drugs

to

serve

another

hospital

Therefore

the

amount

of

drugs

processed

in

was

not

fully

representative

for

the

amount

of

drugs

processed

in

the

years

that

this

site

was

in

use

Site

showed

the

highest

contamination

which

paralleled

the

relative

amount

of

drug

handled

in

this

unit

As

expected

platinum

was

found

in

most

wipe

samples

taken

from

the

middle

of

the

LAF

hood

bench

Notable

was

that

in

general

wipe

samples

of

the

front

edge

of

the

LAF

hood

were

more

contaminated

than

samples

taken

from

the

bench-top

of

the

LAF

hood

Furthermore

floor

samples

usually

contained

the

highest

platinum

levels

Other

locations

showing

substantial

contamination

were

storage

shelves

door

handles

and

handles

of

service

hatches

Duplicate

samples

of

locations

and

showed

similar

results

indicating

a

homogeneous

distribution

over

de

surface

area

The

number

of

years

that

the

seven

units

were

in

use

did

not

parallel

contamination

levels

and

the

amount

of

drug

handled

in

overall

did

not

predict

the

level

of

contamination

either

Discussion

The

presence

of

cytotoxic

drug

contamination

in

hospital

pharmacies

is

recognized

as

a

potential

health

risk

Therefore

it

is

important

to

monitor

this

contamination

Because

platinum

coordination

complexes

belong

to

the

most

extensively

used

anticancer

agents

it

is

relevant

to

focus

on

the

occupational

exposure

of

these

drugs

The

rationale

for

evaluation

of

platinum

contamination

is

also

illustrated

by

several

studies

showing

increased

levels

of

platinum

in

blood

-LRB-

Nygren

and

Lundgren

-RRB-

and

urine

-LRB-

Ensslin

et

al.

Nygren

and

Lundgren

Pethran

et

al.

Schreiber

et

al.

Turci

et

al.

-RRB-

of

hospital

personnel

working

with

these

agents

To

be

able

to

accurately

assess

the

platinum

contamination

originating

from

cisplatinum

oxaliplatinum

and

carboplatinum

at

different

locations

we

developed

and

validated

a

wipe

sampling

method

ICP-MS

was

used

for

quantification

of

platinum

because

this

technique

assures

a

high

sensitivity

and

relative

simple

sample

pre-treatment

The

sensitivity

of

the

method

was

excellent

The

LLOQ

was

set

at

a

platinum

concentration

of

ng

l

corresponding

to

pg

per

sample

or

pg

cm

when

wiping

a

surface

of

To

our

best

knowledge

the

method

described

here

is

times

more

sensitive

than

other

methods

described

for

determination

of

platinum

in

wipe

samples

-LRB-

Connor

et

al.

Mason

et

al.

Raghavan

et

al.

Schmaus

et

al.

Ziegler

et

al.

-RRB-

Sample

pre-treatment

only

involved

surface

sampling

desorption

and

filtration

After

filtration

samples

could

be

analysed

immediately

During

method

development

it

was

shown

that

in

addition

to

tissue

material

and

desorption

solvent

also

the

wipe

solvent

affected

the

recovery

to

a

considerable

extent

This

was

in

contrast

with

results

described

by

Turci

et

al.

-LRB-

-RRB-

who

mentioned

that

the

type

of

wipe

solvent

would

not

influence

the

recovery

since

contaminants

would

be

wept

away

from

the

surfaces

independent

of

the

composition

or

the

pH

of

the

solution

itself

Best

recoveries

were

achieved

by

wiping

with

Kimtech

Science

precision

wipes

moistened

with

μl

water

and

subsequent

desorption

with

HCl

Validation

of

the

method

was

performed

for

the

three

in

oncology

most

prominently

used

platinum

agents

cisplatinum

oxaliplatinum

and

carboplatinum

We

decided

not

to

choose

one

reference

compound

because

the

different

molecular

structures

of

the

platinum

agents

and

as

a

result

the

variable

physical

characteristics

might

possibly

lead

to

a

variation

in

absorption

and

desorption

characteristics

Excellent

reproducibility

-LRB-

imprecision

up

to

-RRB-

and

recoveries

-LRB-

-RRB-

were

demonstrated

with

spiked

tissues

for

all

concentration

levels

and

compounds

Up

to

of

the

initial

amount

of

platinum

added

to

the

tissues

was

not

recovered

after

desorption

and

analysis

This

could

be

due

to

variation

in

analysis

as

well

as

loss

due

to

adsorption

to

the

tissues

Recoveries

from

the

spiked

stainless

steel

surface

were

for

cisplatinum

for

oxaliplatinum

and

for

carboplatinum

Recoveries

for

the

three

compounds

from

the

linoleum

surface

were

respectively

and

These

results

showed

that

for

stainless

steel

depending

on

the

compound

analysed

up

to

of

the

initial

amount

of

platinum

spiked

to

the

surface

was

lost

This

was

for

the

greater

part

caused

by

the

inability

of

the

wipe

procedure

to

remove

all

the

added

platinum

and

for

a

minor

part

by

the

variation

in

analysis

and

loss

due

to

adsorption

to

the

tissues

The

lower

recovery

that

was

observed

for

cisplatinum

is

most

probably

a

consequence

of

its

superior

reactivity

compared

to

oxaliplatinum

and

carboplatinum

This

might

lead

to

a

stronger

binding

affinity

of

cisplatinum

to

materials

and

surfaces

For

linoleum

up

to

of

the

initial

amount

of

platinum

spiked

to

the

surface

was

not

recovered

For

this

surface

recoveries

were

similar

for

all

compounds

To

evaluate

the

stability

of

spiked

samples

recoveries

were

assessed

after

storage

at

room

temperature

and

°C

Storage

of

spiked

tissues

at

°C

for

at

least

weeks

was

possible

However

storage

of

tissues

spiked

with

cisplatinum

for

week

at

room

temperature

led

to

considerable

decrease

in

platinum

levels

Even

though

platinum

concentrations

from

oxaliplatinum

and

carboplatinum

spiked

tissues

did

not

reduce

under

these

conditions

storage

at

room

temperature

was

not

recommended

also

because

the

source

of

elemental

platinum

is

not

known

in

wipe

samples

performed

in

pharmacies

Differences

in

recovery

of

the

cisplatinum

and

the

oxaliplatinum

and

carboplatinum

spiked

samples

again

could

be

explained

by

the

higher

reactivity

of

cisplatinum

For

a

correct

interpretation

of

surface

sampling

results

it

is

relevant

to

take

into

account

that

platinum

is

an

element

that

not

only

appears

in

the

environment

due

to

contamination

with

platinum

containing

cytotoxic

drugs

but

also

due

to

pollution

by

car

exhaust

catalysts

By

wipe

sampling

two

locations

where

no

cytotoxic

drugs

were

handled

we

therefore

determined

that

below

a

threshold

of

ng

l

platinum

-LRB-

pg

cm

when

wiping

a

surface

of

-RRB-

it

was

not

possible

to

address

the

source

of

contamination

All

surfaces

in

the

preparation

units

with

platinum

levels

above

the

threshold

were

considered

as

being

contaminated

by

platinum

containing

drugs

Taking

this

threshold

into

consideration

platinum

contamination

was

reported

in

of

the

samples

taken

in

the

seven

Dutch

hospital

pharmacies

It

is

important

to

consider

that

recoveries

of

the

samples

as

assessed

in

the

validation

study

deviate

from

dependent

on

the

type

of

surface

sampled

and

on

the

type

of

compounds

present

on

the

surface

Therefore

results

depicted

in

Table

represent

of

the

actual

contamination

present

on

the

surface

The

results

of

this

study

indicate

that

there

is

substantial

variation

in

surface

contamination

of

the

pharmacies

tested

and

that

the

amount

of

platinum

processed

in

the

pharmacies

did

not

always

parallel

the

level

of

contamination

The

number

of

preparations

with

platinum

drugs

was

however

not

assessed

and

might

also

be

related

to

surface

contamination

This

suggests

that

variation

in

the

application

of

or

compliance

to

cleaning

and

working

procedures

and

the

incidence

of

calamities

rather

than

the

amount

of

platinum

processed

caused

variation

in

surface

contamination

In

general

results

reveal

that

the

LAF

hoods

the

floor

in

front

of

the

LAF

hoods

door

handles

and

handles

of

service

hatches

were

often

contaminated

This

demonstrates

that

contamination

is

often

spread

throughout

the

pharmacy

Notable

was

that

wipe

samples

of

the

front

edge

of

the

LAF

hood

were

more

contaminated

than

samples

from

the

bench-top

of

the

LAF

hood

This

is

thought

to

be

due

to

incorrect

application

of

working

procedures

or

insufficient

cleaning

By

taking

duplicate

wipe

samples

of

the

LAF

hood

the

front

edge

of

the

LAF

hood

and

the

floor

we

demonstrated

that

these

locations

were

overall

contaminated

and

that

contamination

did

not

appear

to

be

spotty

as

was

mentioned

by

Zeedijk

et

al.

-LRB-

-RRB-

We

also

investigated

storage

shelves

and

in

most

pharmacies

considerable

platinum

contamination

was

found

These

elevated

levels

could

be

a

consequence

of

elevated

levels

of

platinum

on

packaging

material

-LRB-

Connor

et

al.

Mason

et

al.

Nygren

et

al.

-RRB-

Contamination

of

packaging

can

lead

to

a

spread

of

cytotoxic

drugs

to

locations

where

the

drugs

are

stored

or

processed

Therefore

it

was

not

surprising

that

the

storage

shelves

of

platinum

agents

were

contaminated

Furthermore

it

was

noticed

that

in

at

least

one

of

the

hospital

pharmacies

-LRB-

at

site

-RRB-

secondary

packaging

and

caps

of

the

vials

were

discarded

onto

the

floor

during

preparation

most

probably

to

prevent

the

packaging

from

interfering

with

preparation

activities

This

pharmacy

indeed

showed

considerable

contamination

of

the

floor

In

our

study

surfaces

showed

platinum

contamination

of

up

to

pg

cm

The

results

are

in

the

same

range

as

findings

of

some

other

studies

describing

surface

contamination

of

platinum

in

hospital

pharmacies

-LRB-

Leboucher

et

al.

Schmaus

et

al.

-RRB-

In

the

study

of

Leboucher

et

al.

-LRB-

-RRB-

however

no

platinum

was

found

outside

the

LAF

hood

which

could

be

due

to

the

high

detection

limit

of

the

atomic

absorption

spectrometry

method

used

-LRB-

μg

l

-RRB-

Schmaus

et

al.

-LRB-

-RRB-

performed

a

study

in

hospital

pharmacies

and

all

samples

tested

positive

for

platinum

even

though

the

LLOQ

of

their

method

was

eight

times

higher

than

of

the

method

described

here

Yet

although

in

our

study

not

all

samples

tested

positive

for

platinum

the

highest

contamination

found

-LRB-

pg

cm

-RRB-

was

comparable

to

the

highest

contamination

found

by

Schmaus

et

al.

-LRB-

pg

cm

-RRB-

Mason

et

al.

-LRB-

-RRB-

showed

lower

contamination

levels

than

found

in

our

study

despite

that

the

amount

of

drug

handled

in

these

pharmacies

was

higher

than

in

the

pharmacies

in

our

study

When

comparing

the

amounts

of

platinum

detected

on

different

locations

in

this

study

-LRB-

up

to

μg

per

wipe

sample

-RRB-

with

the

platinum

content

of

one

vial

-LRB-

between

and

mg

platinum

-RRB-

contamination

seems

to

be

relatively

low

Furthermore

the

extremely

sensitive

technique

used

in

this

study

leads

to

a

high

percentage

of

positive

samples

Interpretation

of

these

results

is

rather

complicated

It

is

important

to

consider

that

the

total

area

of

the

contamination

is

large

and

that

pharmacy

personnel

are

at

risk

to

be

exposed

to

the

contamination

daily

Hence

for

safety

precautions

it

is

recommended

to

attempt

to

achieve

the

lowest

possible

contamination

Environmental

monitoring

therefore

may

be

used

to

monitor

and

control

contamination

and

thereby

evaluate

working

and

cleaning

procedures

rather

than

to

interpret

potential

health

risks

In

general

when

a

minimal

contamination

level

is

desired

the

results

of

this

study

demonstrate

that

cleaning

and

working

procedures

do

not

sufficiently

prevent

contamination

in

most

hospitals

This

could

be

due

to

an

inadequate

compliance

of

personnel

to

these

procedures

Moreover

contamination

can

spread

out

unconsciously

by

hands

or

feet

of

the

personnel

It

is

also

likely

that

cleaning

procedures

as

applied

in

the

different

pharmacies

are

not

fully

optimised

and

validated

leading

to

contamination

due

to

sub-optimal

cleaning

With

respect

to

the

physical

properties

of

the

different

cytotoxic

drugs

it

is

recommended

to

consider

cleaning

techniques

appropriate

for

specific

agents

As

was

shown

in

this

study

for

platinum

for

instance

ethanol

did

not

effectively

remove

platinum

from

a

stainless

steel

surface

Although

water

gave

better

recoveries

for

platinum

it

was

not

capable

of

removing

all

of

the

added

platinum

from

the

stainless

steel

surface

This

illustrates

the

importance

to

evaluate

several

cleaning

procedures

for

the

different

cytotoxic

agents

handled

and

to

optimise

a

procedure

which

does

remove

all

drugs

with

acceptable

recoveries

In

conclusion

we

developed

and

validated

an

ultra

sensitive

and

reliable

ICP-MS

method

for

the

determination

of

platinum

in

surface

samples

This

method

was

successfully

applied

in

the

evaluation

of

platinum

contamination

in

the

preparation

units

of

seven

Dutch

hospital

pharmacies

It

was

demonstrated

that

pharmacy

personnel

is

at

risk

to

be

exposed

to

platinum

despite

the

use

of

cleaning

and

safety

procedures

As

long

as

the

consequences

of

long-term

exposure

are

not

known

the

aim

should

be

to

achieve

a

contamination

levels

as

low

as

possible

This

study

therefore

highlights

the

need

to

further

evaluate

cleaning

and

safety

procedures

Wipe

sampling

can

be

applied

to

quantify

improvements

made

through

changes

in

procedures

O O B B I 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I 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 O O O O O O O O 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 O O O O O O O O O O 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 O O O O O O O O O O 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 O O O O O O O B O O O O O O O O B I 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 I 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O 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 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 B I O O O B I 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 B I 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 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 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 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 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 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Protein kinase D enzymes are dispensable for proliferation , survival and antigen receptor-regulated NFκB activity in vertebrate B-cells To investigate the importance of protein kinase D -LRB- PKD -RRB- enzymes we generated a PKD-null DT40 B-lymphocyte cell line . Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells -LSB- Matthews , S.A. , Liu , P. , Spitaler , M. , Olson , E.N. , McKinsey , T.A. , Cantrell , D.A. and Scharenberg , A.M. -LRB- 2006 -RRB- Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes . Mol . Cell Biol . 26 , 1569 -- 1577 -RSB- . We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells . However , in contrast to previous observations in other cell types , PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses , nor NFκB transcriptional activity downstream of the B cell antigen receptor . Thus , PKDs have a selective role in DT40 B-cell biology . 1 Introduction The protein kinase D -LRB- PKD -RRB- serine/threonine kinase family has three members : PKD1 , PKD2 and PKD3 . Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells , where they are activated in response to antigen receptors stimulation -LSB- 2,3 -RSB- . A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol -LRB- DAG -RRB- which stimulates classical and/or novel protein kinase Cs -LRB- PKC -RRB- that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases -LSB- 3 -- 6 -RSB- . The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation -LSB- 7 -RSB- as well as regulating the spatial location of PKD enzymes within cells -LSB- 8 -- 12 -RSB- . PKD enzymes have been proposed to regulate numerous cellular functions , including cell proliferation -LSB- 13 -- 16 -RSB- , anti-apoptotic signals -LSB- 17,18 -RSB- and thymocyte development -LSB- 19 -RSB- . Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function , cell adhesion and cell motility -LRB- reviewed in -LSB- 20 -RSB- -RRB- . In particular , PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress -LSB- 17,21 -- 23 -RSB- . Another recently proposed PKD1 substrate is HSP27 -LSB- 24 -RSB- , a small heat shock protein involved in regulating cell migration and cell survival -LSB- 25 -RSB- . An essential role for PKD enzymes in regulating class II histone deacetylases -LRB- HDACs -RRB- , enzymes that repress MEF2-dependent gene transcription , has also been demonstrated -LSB- 1,26 -- 28 -RSB- . To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family -LSB- 1 -RSB- , allowing us to investigate the function -LRB- s -RRB- of PKD isoforms following B cell antigen receptor -LRB- BCR -RRB- stimulation , as well addressing the issue of functional redundancy between the different PKD family members . Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells -LSB- 1 -RSB- . Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells . However , PKD-null DT40 B cells are viable and proliferate normally . Moreover , loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity . Together , these findings reveal that in B lymphocytes , PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems . 2 Materials and methods 2.1 Cell culture , transient transfections and cell stimulation The generation , culture and activation of PKD1 − / − , PKD3 − / − and PKD1/3 − / − knockout DT40 B cell lines have been described previously -LSB- 1 -RSB- . Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described -LSB- 1 -RSB- . Chloramphenicol acetyl transferase assays have been described previously -LSB- 29 -RSB- . 2.2 sIgM staining DT40 B cells -LRB- 2 × 106 cells per point -RRB- were resuspended in 200 μl buffer -LRB- RPMI 1640 media , 1 % foetal calf serum -RRB- containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice . The cells were washed twice and fluorescent intensity was analysed by flow cytometry . All results shown are representative of at two to four independent experiments unless otherwise indicated . 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms , PKD1 and PKD3 , and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes -LSB- 1 -RSB- . In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3 − / − cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter . Hence , in the presence of doxycycline , Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype -LRB- Fig. 1A -RRB- . Previously , we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases -LRB- HDACs -RRB- during BCR engagement is defective in PKD1/3 − / − B cells and can restored upon re-expression of a single PKD isoform -LSB- 1 -RSB- . The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate -LSB- 24 -RSB- and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82 , the proposed PKD1 substrate sequence . We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3 . As shown in Fig. 1B , activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells . BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells -LRB- Fig. 1B -RRB- . However , BCR - and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 -LRB- Fig. 1C -RRB- . Significantly , doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation -LRB- Fig. 1C -RRB- . In contrast , expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR - or phorbol ester-induced HSP27 phosphorylation -LRB- Fig. 1D -RRB- . Hence , PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases . 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival -LRB- reviewed in -LSB- 20 -RSB- -RRB- . To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence -LRB- PKD1/3 − / − : Flag-PKD3 + ve -RRB- or absence -LRB- PKD1/3 − / − -RRB- of doxycycline and monitored exponential growth . As shown in Fig. 2A , PKD1/3 − / − cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation . Furthermore , the viability of PKD1/3 − / − B cells during routine culturing was not significantly different from that of wild-type B cells -LRB- data not shown -RRB- . It was noted that the population doubling time of PKD1/3 − / − cells was slightly slower than that of wild type DT40 cells -LRB- 12.7 ± 2.8 h versus 10.2 ± 0.4 h -RRB- but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3 − / − cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes . Thus , PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells . PKD enzymes , specifically PKD1 and PKD2 , have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast , HeLa and epithelial cell lines -LSB- 17,30 -- 32 -RSB- . We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli . As shown in Fig. 2B , loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli -LRB- H2O2 or serum deprivation -RRB- ; DNA damaging agents -LRB- etoposide or doxorubicin -RRB- ; ER pathway stress due to calcium overload -LRB- thapsigargin -RRB- or following prolonged treatment with phorbol esters or Trichostatin A , an inhibitor of class I/II HDACs . Thus , PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli . 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells . Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3 − / − -LRB- and in PKD1/3 − / − : Flag-PKD3 + ve -RRB- cells compared to wild-type DT40 B cells -LRB- Fig. 3A and data not shown -RRB- . Nevertheless , BCR-crosslinking of PKD1/3 − / − cells was sufficient to induce the activation of a number of signalling cascades , similar to that observed in wild-type cells -LRB- Fig. 3B -RRB- . Hence , BCR-induced activation of the Akt , mTOR/p70 S6 kinase -LRB- as shown by S6 ribosomal protein phosphorylation -RRB- and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells -LRB- Fig. 3B -RRB- . Furthermore , enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells -LRB- data not shown -RRB- . We did observe that the strength of BCR -LRB- but not phorbol ester -RRB- - induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3 − / − B cells compared to wild-type B cells -LRB- Fig. 3B -RRB- . One interpretation of this data is that PKD enzymes may modulate Erk activation . Indeed , PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines -LSB- 33 -- 35 -RSB- . However , BCR-induced Erk phosphorylation was also reduced in PKD1/3 − / − - Flag-PKD3 + B cells -LRB- data not shown -RRB- suggesting that reduced BCR levels on the surface of PKD1/3 − / − -LRB- and PKD1/3 − / − - Flag-PKD3 + -RRB- B cells may itself impact on the strength of activation of this specific intracellular signalling pathway . To search for other potential PKD targets that may show defective regulation in PKD1/3 − / − DT40 B cells , we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes -LRB- LxRxxpS/T -RRB- -LSB- 36 -RSB- . As shown in Fig. 3C , phorbol ester - and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3 − / − cells and is therefore independent of PKD enzymes . However , pretreatment of both wild-type and PKD1/3 − / − DT40 B cells with GF109203X , a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs . Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs . This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2 . However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera . BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival , proliferation and function . In this context , it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor . Thus , PKD-mediated activation of NFκB occurs downstream of a variety of different signals , including mROS/oxidative stress , lysophosphatidic acid and the Bcr-Abl oncogene -LSB- 17,21,23,30,37 -RSB- . Furthermore , expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation -LSB- 38 -RSB- . In B cells , NFκB is known to be regulated via DAG and PKCβ -LSB- 39,40 -RSB- but whether PKDs are key intermediaries for NFκB regulation has not been explored . The data -LRB- Fig. 4A -RRB- show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3 − / − DT40 B cells in response to either phorbol ester or BCR stimulation . In contrast , BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ − / − DT40 B cells -LRB- Fig. 4A -RRB- , although strong activation of PKD kinases -LRB- as assessed by autophosphorylation of PKD1 at S916 -RRB- was observed in the PKCβ − / − cells -LRB- Fig. 4B -RRB- . Thus , PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB . 4 Discussion Protein kinase D serine kinases have been proposed to regulate diverse cellular functions including the phosphorylation and nuclear localisation of class II HDACs and the phosphorylation of HSP27 . It has also been suggested that PKDs act as mitochondrial sensors for oxidative stress and play a role in regulating NFκB transcription factors -LSB- 41 -RSB- . Most of the data about the function of PKDs has come from experiments that ectopically express active or inhibitory PKD mutants or that use RNAi to reduce PKD expression . We have used gene targeting to specifically delete PKD alleles in DT40 chicken B cells and can thus use PKD-null DT40 cells to assess the relative contribution of individual PKD isoforms in class II HDAC control versus oxidative stress responses and NFκB regulation in lymphocytes . We have previously used these PKD-null DT40 cells to define an essential role for PKDs in regulation of class II HDACs , the present report now describes an indispensable role for PKDs in regulating the phosphorylation of HSP27 on serine 82 , a site previously identified as a target for the p38-MAPKAPK2 signalling cascade -LSB- 42 -RSB- . However , studies of PKD-null DT40 cells reveal that PKD family kinases are not essential for oxidative stress survival responses nor are they required for activation of NFκB transcription factors . These latter findings are in striking contrast to previous observations in HeLa and epithelial cell lines where overexpression/RNAi approaches have implicated PKD1/2 in the control of proliferation , survival and NFκB activation -LSB- 20,23 -RSB- . Hence , the present report shows that the proposed roles for PKDs as key sensors that modulate survival pathways in response to oxidative stress and regulate cell survival and proliferation are not ubiquitous and may be restricted to certain cell lineages . Taken together , these data indicate that loss of expression of PKD family members does not globally impact on early BCR-regulated signalling pathways .

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

protein kinase d
pkd
proliferation
survival
nfκb
hsp27

pkd, protein kinase d
pkc, protein kinase c
bcr, b cell antigen receptor