The

CIMT-monitoring

panel

a

two-step

approach

to

harmonize

the

enumeration

of

antigen-specific

T

lymphocytes

by

structural

and

functional

assays

The

interpretation

of

the

results

obtained

from

immunomonitoring

of

clinical

trials

is

a

difficult

task

due

to

the

variety

of

methods

and

protocols

available

to

detect

vaccine-specific

T-cell

responses

This

heterogeneity

as

well

as

the

lack

of

standards

has

led

to

significant

scepticism

towards

published

results

In

February

a

working

group

was

therefore

founded

under

the

aegis

of

the

Association

for

Immunotherapy

of

Cancer

-LRB-

CIMT

-RRB-

in

order

to

compare

techniques

and

protocols

applied

for

the

enumeration

of

antigen-specific

T-cell

responses

Here

we

present

the

results

from

two

consecutive

phases

of

an

international

inter-laboratory

testing

project

referred

to

as

the

CIMT

monitoring

panel

A

total

of

centers

from

six

European

countries

participated

in

the

study

in

which

pre-tested

PBMC

samples

synthetic

peptides

and

PE-conjugated

HLA-tetramers

were

prepared

centrally

and

distributed

to

participants

All

were

asked

to

determine

the

number

of

antigen-specific

T-cells

in

each

sample

using

tetramer

staining

and

one

functional

assay

The

results

of

the

first

testing

round

revealed

that

the

total

number

of

cells

analyzed

was

the

most

important

determinant

for

the

sensitive

detection

of

antigen-specific

T-cells

by

tetramer

staining

Analysis

by

ELISPOT

was

influenced

by

a

combination

of

cell

number

and

a

resting

phase

after

thawing

of

peripheral

blood

mononuclear

cells

Therefore

the

experiments

were

repeated

in

a

second

phase

but

now

the

participants

were

asked

to

change

their

protocols

according

to

the

new

guidelines

distilled

from

the

results

of

the

first

phase

The

recommendations

improved

the

number

of

antigen-specific

T-cell

responses

that

were

detected

and

decreased

the

variability

between

the

laboratories

We

conclude

that

a

two-step

approach

in

inter-laboratory

testing

allows

the

identification

of

distinct

variables

that

influence

the

sensitivity

of

different

T-cell

assays

and

to

formally

show

that

a

defined

correction

to

the

protocols

successfully

increases

the

sensitivity

and

reduces

the

inter-center

variability

Such

two-step

inter-laboratory

projects

could

define

rational

bases

for

accepted

international

guidelines

and

thereby

lead

to

the

harmonization

of

the

techniques

used

for

immune

monitoring

Introduction

In

the

last

two

decades

more

than

clinical

trials

of

different

anti-tumor

vaccines

aiming

to

induce

tumor-specific

immunity

in

cancer

patients

have

been

described

-LSB-

-RSB-

Most

of

these

trials

primarily

assessed

safety

and

immunogenicity

while

reporting

partial

or

complete

clinical

responses

in

a

minority

of

patients

-LSB-

-RSB-

Despite

the

fact

that

the

low

fraction

of

clinical

responders

still

precludes

the

establishment

of

a

direct

correlation

between

clinical

efficacy

and

T-cell

reactivity

it

has

become

clear

from

animal

models

and

clinical

observations

that

naturally-occurring

or

vaccine-induced

or

T-cells

play

an

important

role

in

the

control

and

regression

of

tumors

-LSB-

-RSB-

Therefore

the

number

of

subjects

that

mount

a

vaccine-induced

T-cell

response

as

well

as

the

strength

of

a

detected

T-cell

response

represent

important

surrogate

markers

for

vaccine

efficacy

The

enzyme-linked

immunospot

-LRB-

ELISPOT

-RRB-

assay

-LSB-

-RSB-

staining

with

HLA-peptide

multimers

-LSB-

-RSB-

and

intracellular

cytokine

staining

-LRB-

ICS

-RRB-

-LSB-

-RSB-

are

technologies

used

commonly

for

the

monitoring

of

antigen-specific

immune

responses

For

these

three

assays

a

huge

variety

of

different

protocols

are

available

worldwide

This

heterogeneity

together

with

the

fact

that

the

sensitivity

of

the

individual

protocols

can

vary

significantly

makes

a

comparison

of

the

results

obtained

in

different

trials

a

difficult

task

Moreover

an

increasing

number

of

new

technologies

are

constantly

being

introduced

to

the

field

which

makes

interpretations

even

more

complex

-LSB-

-RSB-

Current

data

and

opinion

support

the

use

of

a

functional

assay

like

the

ELISPOT

or

ICS

in

combination

with

a

phenotyping

assay

like

HLA-multimers

-LSB-

-RSB-

but

recognized

international

standards

for

all

these

methodologies

are

still

lacking

The

main

aim

of

the

CIMT

monitoring

panel

is

to

harmonize

and

optimize

the

monitoring

of

antigen-specific

T-cells

among

the

participating

laboratories

based

on

objective

rationales

with

respect

to

the

testing

procedure

the

analysis

and

the

interpretation

of

results

Important

requirements

for

an

immunological

test

are

sensitivity

applicability

to

large

amounts

of

clinical

material

and

feasibility

at

reasonable

cost

The

results

generated

by

the

tests

should

be

reproducible

and

sensitive

independently

of

the

place

where

they

have

been

performed

After

the

first

meeting

of

the

working

group

a

series

of

inter-laboratory

testing

projects

was

initiated

in

which

individual

laboratories

could

compare

their

performance

express

their

needs

and

exchange

experience

in

order

to

improve

their

local

assays

Here

we

report

the

results

of

the

first

two

phases

of

the

CIMT

monitoring

panel

with

participating

centers

from

six

European

countries

Materials

and

methods

Preparation

and

screening

of

PBMC

samples

Buffy

coats

from

HLA-typed

healthy

volunteers

were

kindly

provided

from

the

Blood

Bank

of

the

University

Mainz

HCMV

sero-status

was

known

PBMC

were

isolated

by

Ficoll

density

gradient

separation

-LRB-

Pharmacia

Uppsala

Sweden

-RRB-

washed

two

times

in

RPMI

-LRB-

GIBCO

BRL

Grand

Island

NY

USA

-RRB-

containing

mM

Hepes

buffer

l-arginine

-LRB-

mg/ml

-RRB-

l-glutamine

-LRB-

mg/ml

-RRB-

penicillin

-LRB-

IU/ml

-RRB-

streptomycin

-LRB-

mg/ml

-RRB-

and

FCS

-LRB-

GIBCO

BRL

-RRB-

counted

and

frozen

at

to

cells

per

cryovial

in

ml

of

FCS

DMSO

at

°C

in

freezing-boxes

filled

with

iso-propanol

After

h

all

cryovials

were

transferred

to

liquid

nitrogen

and

stored

until

distribution

to

the

participating

laboratories

Pre-screening

and

selection

of

the

PBMC

donors

for

influenza

and

CMV

T-cell

reactivities

were

performed

by

a

central

lab

using

the

IFNγ

ELISPOT

assay

following

a

local

protocol

as

described

previously

-LSB-

-RSB-

Five

donors

were

selected

for

the

first

phase

of

the

panel

and

eight

for

the

second

phase

One

HLA-A

donor

was

included

in

each

phase

-LRB-

negative

control

-RRB-

all

other

samples

were

HLA-A

Synthetic

peptides

and

HLA-tetramers

Peptides

were

synthesized

using

standard

Fmoc

chemistry

dissolved

at

mg/ml

in

DMSO

aliquotted

and

stored

at

°C

The

purity

was

checked

by

reverse-phase

HPLC

and

was

found

to

be

Two

known

HLA-A

T-cell

epitopes

were

used

influenza

MP

GILGFVFTL

and

HCMV

NLVPMVATV

-LRB-

-RRB-

Biotinylated

recombinant

HLA-A

monomers

folded

with

the

influenza

MP

or

the

HMCV

peptides

were

produced

essentially

as

described

purified

by

gel

filtration

and

stored

as

aliquots

at

°C

-LSB-

-RSB-

Fluorescent

multimers

were

obtained

by

incubation

with

streptavidin-PE

-LRB-

Molecular

Probes

Leiden

The

Netherlands

-RRB-

then

frozen

as

aliquots

after

addition

of

BSA

and

glycerol

HLA-concentrations

of

influenza-tetramer

and

HCMV-tetramers

were

and

μg/ml

respectively

Both

tetramers

were

checked

by

HPLC

and/or

validated

by

staining

of

a

specific

T-cell

line

-LRB-

Influenza

-RRB-

or

PBMC

from

and

CMV

seronegative

donors

-LRB-

CMV

-RRB-

Such

tetramers

are

stable

at

°C

for

at

least

month

-LRB-

personal

observation

-RRB-

and

participants

were

asked

to

perform

all

tests

within

this

time

period

Participating

centers

Twelve

centers

from

five

European

countries

participated

in

the

first

phase

of

the

monitoring

panel

As

one

of

the

investigators

moved

to

another

institution

during

the

study

a

center

from

a

European

country

was

added

to

the

group

in

the

second

phase

of

the

panel

Participation

in

the

panel

was

open

to

all

interested

laboratories

with

a

focus

on

T-cell

monitoring

independently

of

membership

in

the

Association

for

Immunotherapy

of

Cancer

Reagent

distribution

and

assay

guidelines

Coded

PBMC

samples

synthetic

peptides

and

HLA-A

tetramers

were

shipped

on

dry

ice

to

the

participants

Additionally

guidelines

for

the

two

T-cell

assays

were

distributed

for

each

phase

Phase

A

protocol

for

tetramer

staining

was

included

Briefly

PBMC

per

test

were

transferred

directly

after

thawing

into

one

well

of

a

well

u-bottom

plate

and

washed

in

FACS

buffer

consisting

of

PBS

FCS

mM

EDTA

azide

Incubation

with

μg/ml

HLA-tetramer

was

then

performed

in

FACS

buffer

with

FCS

for

min

at

room

temperature

in

the

dark

After

one

wash

in

FACS

buffer

mAb

for

T-cell

staining

were

added

for

min

at

°C

Finally

cells

were

washed

twice

before

fixing

in

FACS

buffer

containing

formaldehyde

solution

Three

mAb

combinations

were

proposed

alone

plus

or

plus

Each

lab

could

choose

here

the

antibody

clones

fluorescent

dye

and

concentrations

used

Stainings

were

performed

in

duplicate

For

the

functional

assays

synthetic

peptides

were

diluted

at

mg/ml

in

PBS

as

a

stock

solution

Concentrations

in

further

tests

were

μg/ml

left

to

the

choice

of

the

participants

There

were

no

recommendation

which

functional

test

should

be

performed

so

that

each

group

could

choose

the

test

either

routinely

used

or

to

be

implemented

for

its

own

needs

In

this

first

phase

laboratories

chose

the

IFNγ

ELISPOT

assay

one

lab

-LRB-

-RRB-

a

FACS-based

intracellular

IFNγ

staining

and

one

lab

performed

both

assays

-LRB-

-RRB-

Spot

counting

was

performed

locally

Phase

Following

the

results

obtained

in

the

first

testing

phase

requirements

were

introduced

and

participants

were

asked

to

apply

exactly

these

new

criteria

-LRB-

two

for

the

tetramer

staining

and

four

for

the

ELISPOT

see

Results

section

-RRB-

The

assay

guidelines

were

modified

accordingly

However

in

order

to

reduce

the

variability

in

the

FACS

analysis

of

the

laboratories

a

figure

showing

exemplary

dot-plots

settings

of

gates

and

quadrants

and

statistical

analysis

was

provided

All

laboratories

were

now

required

to

perform

an

IFNγ

ELISPOT

as

the

functional

test

with

a

fixed

peptide

concentration

of

μg/ml

Participants

were

encouraged

to

use

a

distributed

model

protocol

but

were

allowed

to

use

their

local

protocol

provided

that

they

applied

the

four

new

requirements

introduced

in

the

second

phase

Collection

and

analysis

of

results

After

performing

the

required

tests

in

each

phase

participants

returned

a

completed

report

form

containing

all

relevant

information

Number

of

cells

recovered

after

thawing

was

included

to

assess

viability

after

transport

For

the

tetramer

staining

experiments

mAb

clone

manufacturer

amount

cytometer

type

and

number

of

lymphocytes

and/or

cells

analyzed

were

noted

Results

were

expressed

as

percentage

of

tetramer-positive

cells

among

or

lymphocytes

depending

on

which

mAb

combination

was

used

for

the

staining

Additionally

FACS

dot-plots

containing

all

gates

quadrants

and

deduced

statistical

analysis

were

collected

For

the

functional

test

medium

and

thawing

procedure

-LRB-

e.g.

addition

of

DNAse

of

a

resting

phase

etc.

-RRB-

had

to

be

described

as

well

as

the

number

of

cells

per

test

the

antibodies

used

-LRB-

clone

manufacturer

concentration

-RRB-

the

final

peptide

concentration

and

the

incubation

times

For

the

ELISPOT

assay

the

type

of

plate

the

enzymatic

visualization

system

and

the

spot

reader

were

also

noted

Absolute

spot

numbers

were

given

by

each

participant

and

filter

plates

were

kept

for

possible

second

analysis

All

results

from

both

phases

were

collected

and

centrally

analyzed

For

the

tetramer

stainings

the

number

of

lymphocytes

number

of

T-cells

and

frequencies

of

tetramer-positive

cells

were

calculated

on

the

basis

of

the

stainings

and

statistics

provided

by

the

participants

Apart

from

these

calculated

frequencies

a

visual

evaluation

was

necessary

-LRB-

see

Results

-RRB-

For

the

ELISPOT

analysis

was

performed

based

on

the

spot

numbers

reported

by

the

participants

followed

by

a

student

t

test

Results

were

accepted

as

positive

reaction

only

when

the

numbers

of

antigen-specific

spots

exceeded

the

number

of

spots

in

the

background

wells

by

atleast

a

factor

two

The

coefficients

of

variation

-LRB-

CV

-RRB-

were

calculated

for

all

results

-LRB-

CV

SD/mean

-RRB-

and

are

shown

in

supplementary

Tables

b

The

raw

data

from

both

panel

phases

will

be

provided

to

interested

readers

upon

request

Results

Phase

of

the

interlaboratory

testing

project

general

aspects

Coded

PBMC

samples

from

four

HLA-A

and

one

HLA-A

healthy

donor

-LRB-

-RRB-

were

included

in

this

first

testing

phase

The

thawing

procedure

for

PBMC

samples

in

the

test

centers

was

not

standardized

and

the

recovery

of

viable

cells

varied

greatly

between

and

-LRB-

mean

-RRB-

in

the

labs

However

the

number

of

cells

recovered

was

in

all

cases

sufficient

to

perform

the

required

analyses

When

all

the

data

from

the

tetramer

staining

and

functional

tests

were

combined

it

became

clear

that

subjects

and

had

responded

to

the

HLA-A

restricted

CMV-derived

peptide

consistent

with

their

CMV

seropositive-status

and

that

subjects

and

had

responded

to

influenza

In

total

each

laboratory

should

in

theory

have

been

able

to

measure

six

positive

-LRB-

CMV

and

influenza

-RRB-

responses

Detection

of

antigen-specific

T-cells

by

tetramer

staining

and

IFNγ

ELISPOT

The

protocol

required

that

all

PBMC

samples

should

be

analyzed

by

the

participants

for

the

presence

of

HLA-A

CMV-specific

and

influenza-specific

T-cells

using

centrally-prepared

tetramers

The

indicated

frequencies

of

antigen-specific

cells

generally

represent

the

mean

of

two

separate

stainings

with

Ab/tetramer

except

for

centers

-LRB-

-RRB-

-LRB-

tetramer

-RRB-

and

-LRB-

one

staining

and

one

staining

-RRB-

and

are

based

on

the

analysis

and

dot

plots

provided

by

each

participant

As

illustrated

in

Fig.

the

absolute

numbers

of

tetramer-positive

T-cells

were

influenced

by

the

individual

decision

of

where

to

set

the

gates

and

quadrant

markers

for

the

analysis

For

example

the

inclusion

of

the

subset

of

T

lymphocytes

expressing

at

a

low

density

influenced

the

number

of

and

consequently

the

frequency

of

tetramer

cells

Moreover

non-specific

binding

of

the

tetramer

-LRB-

as

seen

on

the

subset

-RRB-

also

varied

between

the

different

laboratories

For

these

reasons

not

only

the

frequencies

but

also

the

appearance

of

the

tetramer-positive

populations

was

carefully

examined

Two

parameters

were

chosen

for

validation

of

positive

results

-LRB-

-RRB-

a

clustered

but

not

diffuse

tetramer

binding-population

and

-LRB-

-RRB-

strong

intensity

of

tetramer

staining

especially

marked

for

the

CMV-tetramer-binding

population

-LRB-

Fig.

-RRB-

Table

shows

-LRB-

I

-RRB-

the

minimum

mean

and

maximum

frequencies

of

antigen-specific

T-cells

-LRB-

II

-RRB-

the

results

obtained

from

the

individual

centers

and

-LRB-

III

-RRB-

the

number

and

percentage

of

centers

that

detected

a

response

The

high

frequencies

of

CMV-specific

T-cells

in

donors

and

were

readily

detected

by

all

participants

-LRB-

mean

of

per

in

and

mean

of

per

in

respectively

-RRB-

For

influenza-specific

T-cells

the

results

were

more

variable

Influenza-tetramer

cells

in

donor

were

detected

by

all

participants

with

a

mean

frequency

of

one

cell

in

T-cells

In

Donor

of

laboratories

detected

a

mean

of

one

tetramer

binding

cell

per

T-cells

Influenza-specific

cells

were

less

numerous

in

healthy

subjects

and

and

were

only

detected

by

five

and

eight

laboratories

respectively

No

false

positive

reactivity

was

reported

by

any

of

the

participants

Fig.

of

tetramer

staining

results

as

provided

by

four

selected

participating

centers

and

All

stainings

were

performed

on

donor

from

phase

who

showed

reactivity

with

both

of

the

tested

tetramers

Cells

were

gated

either

on

the

lymphocyte

population

-LRB-

-RRB-

or

the

subsets

of

-LRB-

-RRB-

or

-LRB-

-RRB-

according

to

the

Ab

combination

used

by

each

lab

The

upper

panel

shows

results

for

tests

with

the

CMV-tetramer

the

lower

panel

shows

results

for

tests

with

the

influenza-tetramer

In

all

dot-plots

the

tetramer

staining

is

displayed

on

the

y-axis

and

on

the

x-axis

Number

of

counted

T-cells

and

percentage

of

tetramer-positive

cells

among

the

subset

are

indicatedTable

of

the

tetramer

results

from

phase

of

the

CIMT

monitoring

-LRB-

I

-RRB-

-LRB-

II

-RRB-

d

-LRB-

III

-RRB-

Detected

-LRB-

I

-RRB-

Minimum

mean

and

maximum

frequencies

of

antigen-specific

T-cells

-LRB-

II

-RRB-

Results

obtained

from

the

individual

centers

All

frequencies

are

indicated

as

per

x

counted

T-cells

-LRB-

III

-RRB-

Number

and

percentage

of

centers

which

detected

a

given

reactivity

in

donors

and

Eleven

laboratories

analyzed

the

five

PBMC

samples

for

the

presence

of

HLA-A

CMV-specific

and

influenza-specific

IFNγ-producing

T-cells

by

ELISPOT

assay

Only

one

group

-LRB-

-RRB-

used

an

intracellular

cytokine

staining

as

a

functional

test

-LRB-

data

not

shown

because

no

comparison

with

other

groups

possible

-RRB-

Table

shows

-LRB-

I

-RRB-

the

minimum

mean

and

maximum

frequencies

of

antigen-specific

cells

-LRB-

II

-RRB-

the

results

obtained

from

the

individual

centers

and

-LRB-

III

-RRB-

the

number

and

percentage

of

centers

that

detected

each

reactivity

As

described

in

the

Materials

and

methods

results

of

spot-forming

cells

per

seeded

PBMC

were

accepted

as

a

positive

reaction

only

when

passing

statistical

testing

and

when

the

number

of

antigen-specific

spots

exceeded

the

number

of

spots

in

the

background

wells

by

at

least

a

factor

of

two

IFNγ-producing

cells

reactive

against

CMV

were

detected

by

of

the

laboratories

in

donor

-LRB-

mean

reactivity

was

per

PBMC

-RRB-

but

only

by

of

in

donor

-LRB-

mean

reactivity

was

per

PBMC

-RRB-

The

influenza-specific

T-cells

present

in

subject

were

detected

by

six

laboratories

while

the

responses

in

the

healthy

subjects

with

markedly

lower

numbers

of

peripheral

specific

T-cells

-LRB-

and

-RRB-

were

detected

by

three

laboratories

only

Table

of

the

IFNγ

ELISPOT

results

from

phase

of

the

CIMT

monitoring

-LRB-

I

-RRB-

-LRB-

II

-RRB-

Detected

-LRB-

III

-RRB-

Detected

-LRB-

I

-RRB-

Minimum

mean

and

maximum

frequencies

of

antigen-specific

cells

-LRB-

II

-RRB-

Results

obtained

from

the

individual

centers

All

frequencies

are

indicated

as

per

x

seeded

PBMC

except

for

where

it

is

indicated

as

per

x

seeded

T-cellsResults

from

were

not

included

for

calculation

of

the

mean

frequency

of

antigen-specific

T-cells

in

and

-LRB-

III

-RRB-

Number

and

percentage

of

centers

that

detected

a

given

response

in

donors

and

not

determined

Subgroup

analysis

reveals

that

the

number

of

T-lymphocytes

analyzed

affects

the

sensitivity

of

the

tetramer

staining

Although

the

tetramer

stainings

were

performed

with

centrally

prepared

reagents

following

set

guidelines

centers

were

left

free

to

select

several

parameters

according

to

their

own

protocols

and

this

could

have

influenced

the

test

results

-LRB-

see

Materials

and

methods

-RRB-

Most

of

the

participants

used

monoclonal

antibodies

specific

for

and

to

co-stain

the

cells

There

were

no

obvious

differences

in

the

performance

of

the

centers

depending

on

which

antibody

clones

antibody

combinations

or

cytometer

were

used

-LRB-

data

not

shown

-RRB-

There

was

a

high

degree

of

variability

in

the

number

of

cells

which

were

analyzed

per

staining

ranging

from

only

to

about

-LRB-

inter-center

variation

-RRB-

In

addition

a

non-negligible

intra-center

variation

was

observed

for

the

number

of

counted

We

therefore

analyzed

each

individual

staining

independently

of

the

center

that

performed

it

and

focused

on

the

number

of

T-cells

that

had

been

counted

For

the

six

different

antigen-specific

populations

detectable

a

total

of

tests

was

performed

by

the

group

-LRB-

see

Table

-RRB-

Overall

antigen-specific

T-cell

reactivities

were

reported

in

of

the

tests

-LRB-

mean

of

duplicate

stainings

-RRB-

When

less

than

T-cells

were

counted

only

of

all

responses

were

found

In

contrast

of

all

responses

were

manifest

when

more

than

T-cells

were

counted

-LRB-

Fig.

-RRB-

When

antigen-specific

T-cells

were

present

at

high

frequency

the

number

of

cells

counted

did

not

influence

the

result

because

CMV-specific

T-cells

from

donors

and

were

detected

irrespective

of

the

number

of

T-cells

in

the

test

However

for

the

influenza-specific

cells

positivity

was

registered

in

only

of

all

tests

performed

-LRB-

of

tests

-RRB-

Strikingly

we

observed

a

marked

difference

for

the

results

derived

from

those

tests

involving

less

than

T-cells

-LRB-

success

in

detection

-RRB-

as

compared

to

tests

performed

with

more

than

T-cells

-LRB-

-RRB-

Fig.

Subgroup

analysis

of

tetramer

results

from

phase

Bars

indicate

the

percentage

of

positives

that

could

be

detected

by

tetramer

staining

The

first

group

of

bars

shows

the

results

for

all

of

the

six

detectable

positives

the

second

group

shows

results

from

stainings

with

the

CMV-tetramer

and

the

third

group

of

columns

shows

results

from

stainings

with

the

influenza-tetramer

The

open

bars

in

each

group

represent

all

tests

performed

grey

bars

represent

results

obtained

in

tests

that

were

performed

on

more

than

T-cells

and

black

bars

represent

results

obtained

in

tests

that

were

performed

on

less

than

T-cells

The

boxes

within

each

bar

indicate

the

fraction

of

tests

with

a

positive

result

The

asterisk

indicates

a

P-value

by

Chi-square

analysis

b

Subgroup

analysis

of

ELISPOT

results

from

phase

The

bars

indicate

the

percentage

of

positive

reactivities

detected

by

IFNγ

ELISPOT

assays

The

open

bar

shows

the

percentage

of

all

reactivities

detected

by

all

centers

that

performed

the

ELISPOT

assay

as

the

functional

test

Criteria

for

division

of

centers

into

two

subgroups

were

based

on

the

following

requirements

do

not

use

allo-APC

-LRB-

first

subgroup

analysis

-RRB-

use

a

resting

time

-LRB-

second

subgroup

analysis

-RRB-

or

use

equal

or

more

than

PBMC

per

well

-LRB-

third

subgroup

analysis

-RRB-

Grey

bars

always

represent

centers

that

were

in

conformity

with

the

indicated

minimum

requirement

black

bars

show

results

from

centers

that

did

not

fulfil

that

requirement

The

boxes

within

each

column

indicate

the

fraction

of

centers

in

each

category

The

asterisks

indicate

a

P-value

in

Chi-square

analysis

In

conclusion

the

ability

to

detect

antigen-specific

T-cell

reactivities

by

tetramer

staining

was

mainly

affected

by

the

number

of

T-cells

stained

and

analyzed

especially

when

the

antigen-specific

T-cells

were

present

at

low

or

moderate

frequencies

We

therefore

modified

our

guidelines

for

the

tetramer

assay

and

recommended

staining

at

least

PBMC

and

analyzing

all

cells

in

the

tube

In

addition

we

provided

an

example

of

how

optimal

cell

gates

and

dot-plot

quadrants

could

be

selected

ELISPOT

assays

are

heterogeneous

and

require

standardization

The

ELISPOT

analyses

were

performed

according

to

more

or

less

different

protocols

The

most

discernible

differences

that

were

observed

in

these

protocols

concerned

-LRB-

-RRB-

the

different

types

of

multi-screen

plates

-LRB-

-RRB-

the

serum

origin

-LRB-

-RRB-

the

use

of

duplicates

triplicates

or

quadruplicates

-LRB-

-RRB-

the

use

of

allogeneic

APC

-LRB-

-RRB-

the

inclusion

of

a

resting

phase

after

thawing

the

PBMC

-LRB-

-RRB-

the

number

of

PBMC

per

well

-LRB-

-RRB-

the

type

of

antibodies

used

-LRB-

-RRB-

the

type

of

spot-reader

and

the

-LRB-

-RRB-

enzyme

and

substrate

for

staining

of

the

spots

Each

center

also

used

a

different

plate

protocol

-LRB-

distribution

of

the

wells

number

of

replicates

control

tests

-RRB-

The

influence

of

each

of

these

parameters

on

the

number

of

positive

responses

was

studied

by

further

analysis

in

which

the

laboratories

were

divided

into

two

subgroups

As

a

result

several

criteria

were

identified

which

could

help

to

improve

the

sensitivity

and

comparability

of

detection

All

data

sets

-LRB-

duplicates

triplicates

or

quadruplicates

-RRB-

were

first

analyzed

by

Student

t

test

for

unpaired

samples

-LRB-

Materials

and

methods

-RRB-

In

our

panel

one

center

used

quadruplicates

nine

centers

used

triplicates

and

one

center

performed

the

ELISPOT

analysis

in

duplicates

Due

to

the

variety

in

the

replicates

responses

measured

by

duplicate

wells

failed

to

pass

the

Student

t

test

more

often

as

compared

to

triplicates

Overall

the

centers

were

able

to

detect

of

all

possible

reactivities

in

this

panel

phase

-LRB-

Table

Fig.

-RRB-

In

a

subgroup

of

three

laboratories

-LRB-

and

-RRB-

an

allogeneic

APC

population

-LRB-

or

cells

-RRB-

was

added

for

binding

and

presentation

of

the

synthetic

peptides

The

three

centers

that

used

allo-APC

detected

only

of

all

responses

while

the

other

centers

detected

of

all

responses

In

five

laboratories

-LRB-

and

-RRB-

PBMC

were

thawed

and

then

incubated

in

culture

medium

at

°C

After

this

resting

phase

of

h

living

cells

were

washed

counted

and

seeded

into

ELISPOT

plates

Laboratories

using

a

resting

phase

detected

of

the

positive

reactivities

-LRB-

out

of

potentially

positive

tests

-RRB-

No

significant

difference

in

the

ability

to

detect

antigen-specific

T

cells

was

found

using

shorter

or

longer

resting-times

In

contrast

the

laboratories

that

did

not

use

a

resting

procedure

detected

only

of

all

positives

-LRB-

Fig.

-RRB-

Finally

the

number

of

cells

seeded

per

well

differed

considerably

between

all

participants

and

ranged

from

to

PBMC

We

divided

the

laboratories

arbitrarily

into

two

groups

those

using

either

more

than

PBMC

-LRB-

and

-RRB-

or

less

than

PBMC

-LRB-

and

-RRB-

The

first

group

detected

of

all

positive

samples

whereas

the

second

group

was

able

to

detect

only

of

all

positives

-LRB-

Fig.

-RRB-

Centers

and

used

a

defined

number

of

separated

T-cells

in

the

ELISPOT

and

were

therefore

not

included

in

this

subgroup

analysis

other

of

the

nine

depicted

protocol

variables

had

any

obvious

impact

on

the

detection

of

specific

T-cells

As

a

conclusion

from

these

results

four

minimum

requirements

were

formulated

for

the

ELISPOT

protocol

-LRB-

-RRB-

perform

triplicates

for

each

test

antigen

-LRB-

-RRB-

do

not

use

allo-APC

-LRB-

-RRB-

add

a

resting

time

to

increase

the

proportion

of

living

cells

seeded

and

-LRB-

-RRB-

use

a

minimum

number

of

PBMC

per

well

Phase

of

the

interlaboratory

testing

project

general

aspects

To

formally

prove

that

the

requirements

formulated

for

tetramer

staining

and

ELISPOT

analysis

increase

the

ability

of

the

participants

to

detect

antigen-specific

T-cells

and

reduce

the

inter-center

variability

we

decided

to

repeat

the

analysis

in

a

second

phase

of

the

panel

with

the

same

participants

-LRB-

phase

-RRB-

In

this

round

all

groups

were

asked

to

follow

our

modified

guidelines

for

the

tetramer

and

the

ELISPOT-assays

Again

all

PBMC

samples

were

prepared

and

pre-tested

in

one

central

lab

and

peptide

antigens

and

PE-conjugated

tetramers

were

also

provided

from

one

source

As

one

investigator

had

meanwhile

moved

to

another

lab

we

added

a

center

to

the

group

PBMC

from

seven

selected

healthy

HLA-A

donors

and

HLA-A

donor

-LRB-

-RRB-

were

required

to

be

analyzed

for

the

presence

of

HLA-A

CMV-specific

T

cells

and

for

influenza-specific

T-cells

The

mean

number

of

recovered

cells

after

thawing

was

sufficient

to

perform

the

tests

When

all

the

data

were

combined

it

became

clear

that

subjects

and

possessed

CMV-specific

T-cell

subsets

and

and

possessed

influenza-specific

T-cells

Therefore

each

laboratory

could

theoretically

have

measured

eight

positives

-LRB-

CMV

and

Influenza

-RRB-

in

this

second

phase

Analysis

of

T-cell

tetramer

binding

using

the

new

guidelines

In

the

second

phase

a

total

of

tests

were

performed

to

detect

the

eight

possible

tetramer

reactivities

Following

the

modified

guidelines

for

tetramer

staining

the

mean

number

of

T-cells

that

were

counted

in

each

separate

test

increased

markedly

-LRB-

-RRB-

a

mean

of

about

cells

were

analyzed

in

the

phase

I

-LRB-

n

tests

-RRB-

and

a

mean

of

T-cells

in

phase

II

-LRB-

n

tests

-RRB-

The

number

of

cells

per

test

ranged

from

to

In

-LRB-

of

-RRB-

of

the

tests

were

counted

-LRB-

compared

to

of

all

relevant

tests

in

the

first

phase

-RRB-

Table

shows

-LRB-

I

-RRB-

the

minimum

mean

and

maximum

frequencies

of

antigen-specific

T-cells

-LRB-

II

-RRB-

the

results

obtained

from

the

individual

centers

and

-LRB-

III

-RRB-

the

number

and

percentage

of

centers

that

detected

each

T-cell

specificity

Donors

and

showed

very

strong

reactivities

with

the

CMV-tetramer

with

mean

frequencies

of

T-cells

T-cells

and

T-cells

respectively

All

laboratories

were

able

to

detect

these

populations

-LRB-

Table

-RRB-

All

but

one

center

detected

the

influenza-specific

cells

present

at

high

frequencies

in

donors

-LRB-

T-cells

-RRB-

and

-LRB-

T-cells

-RRB-

Donors

and

possessed

fewer

specific

cells

-LRB-

and

T-cells

-RRB-

which

were

found

by

and

centers

respectively

Three

laboratories

also

reported

influenza

tetramer-binding

cells

in

or

According

to

the

results

of

the

other

centers

as

well

as

from

the

ELISPOT

-LRB-

see

below

-RRB-

these

stainings

were

considered

as

false

positive

-LRB-

not

shown

-RRB-

One

center

-LRB-

-RRB-

was

not

able

to

detect

any

of

the

influenza-specific

T-cell

reactivities

Finally

no

tetramer

cells

were

described

in

the

HLA-A

donor

-LRB-

-RRB-

Table

of

tetramer

results

from

phase

of

the

CIMT

monitoring

-LRB-

I

-RRB-

-LRB-

II

-RRB-

-LRB-

III

-RRB-

Detected

-LRB-

I

-RRB-

Minimum

mean

and

maximum

frequencies

of

antigen-specific

T-cells

-LRB-

II

-RRB-

Results

obtained

from

the

individual

centers

All

frequencies

are

indicated

as

per

x

counted

T-cells

-LRB-

III

-RRB-

Number

and

percentage

of

centers

that

detected

each

of

the

eight

possible

responses

Analysis

of

T-cell

responses

by

ELISPOT

following

the

introduction

of

a

set

of

four

rules

In

this

second

phase

all

laboratories

performed

ELISPOT

analysis

following

local

protocols

all

of

which

conformed

to

the

newly

introduced

minimum

requirements

Table

shows

-LRB-

I

-RRB-

the

minimum

mean

and

maximum

frequencies

of

antigen-specific

cells

-LRB-

II

-RRB-

the

results

obtained

from

the

individual

centers

and

-LRB-

III

-RRB-

the

number

and

percentage

of

centers

that

detected

the

response

High

frequency

T-cell

responses

against

CMV

could

readily

be

detected

by

all

centers

in

donors

and

and

by

of

in

donor

Failure

of

center

to

detect

the

CMV

reactivity

in

donor

was

due

to

a

very

high

background

of

the

medium

control

The

number

of

spots

representing

IFNγ-producing

cells

after

influenza-peptide

stimulation

was

generally

lower

and

consequently

the

influenza-specific

T-cell

responses

in

subjects

and

were

detected

by

fewer

laboratories

-LRB-

four

centers

for

three

centers

for

two

centers

for

and

ten

centers

for

-RRB-

The

high

numbers

of

influenza-specific

T-cells

present

in

were

detected

by

all

laboratories

-LRB-

Table

-RRB-

Table

of

IFNγ

ELISPOT

results

from

phase

of

the

CIMT

monitoring

,1181,79133,80358,8241,74548,38714,7201,698

-LRB-

I

-RRB-

,8231,08816,39549,96099914,2654,6691,023

-LRB-

II

-RRB-

Detected

-LRB-

III

-RRB-

Detected

-LRB-

I

-RRB-

Minimum

mean

and

maximum

frequencies

of

antigen-specific

cells

-LRB-

II

-RRB-

Results

obtained

from

the

individual

centers

All

frequencies

are

indicated

as

per

x

seeded

PBMC

-LRB-

III

-RRB-

Number

and

percentage

of

centers

that

detected

each

of

the

eight

possible

responses

Comparison

of

the

results

obtained

in

both

phases

When

the

mean

frequencies

of

all

T-cell

responses

in

both

testing

rounds

were

compared

it

became

clear

that

there

was

a

difference

in

the

distribution

of

reactivities

-LRB-

Fig.

-RRB-

In

the

tetramer

assay

the

mean

T-cell

frequency

of

the

six

possible

positives

in

the

first

phase

was

per

T-cells

This

value

was

per

T-cells

for

the

eight

possible

positives

in

the

second

phase

Similarly

the

mean

T-cell

frequency

of

the

responses

detected

in

IFNγ

ELISPOT

was

per

PBMC

for

Phase

but

per

PBMC

for

Phase

To

allow

a

comparison

of

the

overall

performance

in

both

phases

of

the

panel

we

therefore

decided

to

define

theoretical

thresholds

for

high

moderate

and

low

T-cell

responses

and

then

to

compare

data

of

the

participating

laboratories

within

these

groups

Fig.

of

antigen-specific

T-cell

frequencies

in

the

two

testing

phases

as

obtained

by

tetramer

staining

-LRB-

a

-RRB-

and

IFNγ

ELISPOT

assays

-LRB-

b

-RRB-

The

figure

shows

the

six

reactivities

-LRB-

filled

circle

-RRB-

and

the

calculated

mean

of

all

reactivities

from

phase

-LRB-

filled

line

-RRB-

as

well

as

the

eight

reactivities

-LRB-

open

circle

-RRB-

and

calculated

mean

of

all

reactivities

from

phase

-LRB-

open

line

-RRB-

The

frequency

of

antigen-specific

T-cells

is

indicated

on

the

y-axis

as

per

x

counted

T-cells

for

the

tetramer

test

and

as

per

x

seeded

PBMC

for

the

ELISPOT

assay

In

order

to

define

such

thresholds

for

low

medium

and

high

T-cell

responses

we

first

displayed

the

probability

of

detecting

each

of

the

different

reactivities

as

a

value

in

a

coordinate

system

and

inserted

a

trendline

For

both

the

tetramer

assay

and

the

ELISPOT

assay

we

observed

a

clear

correlation

between

the

frequencies

of

antigen-specific

T-cells

and

the

number

of

participating

centers

that

were

able

to

detect

these

populations

We

then

calculated

the

theoretical

frequencies

at

which

-LRB-

y

-RRB-

and

-LRB-

y

-RRB-

of

all

participants

could

detect

a

given

response

-LRB-

Fig.

b

-RRB-

and

used

these

two

thresholds

to

divide

all

reactivities

into

three

distinct

classes

of

T-cell

responses

-LRB-

high

moderate

and

low

-RRB-

Fig.

of

detecting

a

reactivity

by

a

tetramer

staining

or

b

IFNγ

ELISPOT

assay

A

trendline

was

inserted

on

the

basis

of

results

from

all

reactivities

from

both

phases

of

the

panel

The

figure

shows

the

six

reactivities

from

phase

-LRB-

filled

squares

-RRB-

and

the

eight

reactivities

from

phase

-LRB-

open

squares

-RRB-

The

frequency

of

antigen-specific

T-cells

is

shown

on

the

x-axis

in

per

x

counted

T-cells

for

the

tetramer

assay

-LRB-

a

-RRB-

or

per

x

seeded

PBMC

for

the

ELISPOT

assay

-LRB-

b

-RRB-

X-values

for

y

and

y

are

indicated

by

the

broken

lines

For

the

tetramer

assay

T-cell

frequencies

exceeding

per

T-cells

were

therefore

classified

as

high

whereas

frequencies

of

less

than

per

were

classified

as

low

-LRB-

Fig.

-RRB-

Following

the

same

rules

for

the

ELISPOT

assay

T-cell

responses

of

at

least

one

IFNγ

spot

per

PBMC

can

be

considered

as

high

and

T-cell

responses

of

less

than

one

spot

per

PBMC

as

low

-LRB-

Fig.

-RRB-

With

these

calculated

assay-specific

thresholds

for

high

moderate

and

low

T-cell

responses

we

compared

the

results

obtained

in

the

two

phases

For

the

tetramer

assay

the

ability

to

detect

high

frequency

T-cells

-LRB-

per

-RRB-

did

not

differ

in

the

two

phases

and

was

not

influenced

by

the

number

of

analyzed

as

previously

seen

for

each

of

the

two

phases

separately

-LRB-

Fig.

-RRB-

However

for

moderate

and

low

T-cell

frequencies

we

found

that

they

could

be

successfully

detected

in

only

of

cases

in

the

first

phase

but

this

improved

to

in

the

second

phase

Moreover

here

the

number

of

cells

counted

did

have

an

impact

on

the

ability

to

detect

low

frequency

T-cells

In

the

first

phase

only

were

detected

when

less

than

were

counted

as

compared

to

when

more

than

T-cells

were

counted

The

same

trend

was

observed

in

phase

but

in

this

case

of

assays

with

less

than

successfully

detected

the

moderate

to

low

T-cell

frequencies

compared

to

counting

more

than

-LRB-

Fig.

-RRB-

Fig.

Percentage

of

reactivities

actually

detected

by

tetramer

staining

The

first

two

groups

of

bars

show

the

detection

rate

for

the

nine

high

reactivities

-LRB-

per

T-cells

-RRB-

in

phase

and

phase

The

next

two

groups

of

bars

show

the

detection

rate

for

five

moderate

to

low

reactivities

-LRB-

per

-RRB-

in

phase

-LRB-

third

group

-RRB-

or

phase

-LRB-

fourth

group

-RRB-

The

open

bars

represent

all

tests

performed

grey

bars

represent

results

obtained

in

tests

that

were

performed

on

more

than

T-cells

and

filled

bars

represent

results

obtained

in

tests

that

were

performed

on

less

than

T-cells

b

Percentage

of

reactivities

detected

in

IFNγ

ELISPOT

assays

The

first

two

groups

of

bars

show

the

rate

of

detection

of

the

four

high

reactivities

-LRB-

per

PBMC

in

phase

and

phase

The

next

two

groups

of

columns

show

the

rate

of

detection

for

the

ten

moderate

to

low

reactivities

-LRB-

per

PBMC

-RRB-

in

phase

and

phase

The

open

bars

represent

the

performances

of

all

centers

in

the

respective

panel

phase

grey

bars

represent

results

obtained

from

the

five

centers

that

already

fulfilled

at

least

three

of

the

four

minimum

criteria

in

phase

and

filled

bars

represent

results

obtained

from

centers

that

fulfilled

less

than

three

of

the

four

minimum

criteria

in

phase

We

then

analyzed

the

capacity

of

the

laboratories

to

measure

either

high

T-cell

responses

-LRB-

per

PBMC

-RRB-

or

low

to

moderate

T-cell

responses

-LRB-

per

PBMC

-RRB-

in

the

ELISPOT

assay

This

analysis

was

performed

for

two

defined

subgroups

of

participants

The

first

subgroup

included

those

five

centers

-LRB-

and

-RRB-

that

already

fulfilled

three

or

four

of

the

requirements

in

the

first

phase

of

the

panel

These

five

centers

did

not

have

to

introduce

any

change

or

at

least

no

major

changes

to

their

protocol

for

the

repetition

of

the

experiments

in

phase

II

The

second

subgroup

included

the

new

center

-LRB-

led

by

a

colleague

that

had

been

in

a

laboratory

that

only

fulfilled

one

of

four

requirements

in

phase

I

-RRB-

and

all

others

that

had

fulfilled

only

one

or

two

of

the

four

requirements

in

the

first

phase

All

laboratories

in

this

second

group

had

to

introduce

marked

changes

to

their

locally

established

protocols

Similar

to

the

tetramer

analysis

the

new

requirements

were

not

necessary

to

detect

antigen-specific

responses

among

the

category

of

high

T-cell

frequencies

in

either

the

first

or

second

phases

-LRB-

Fig.

-RRB-

However

applying

the

set

of

rules

defined

in

phase

I

markedly

improved

the

capacity

of

centers

to

detect

the

low

to

moderate

T-cell

responses

The

first

subgroup

detected

a

total

of

of

the

low

to

moderate

reactivities

in

phase

I

whereas

the

second

subgroup

detected

only

-LRB-

Fig.

-RRB-

After

harmonization

of

the

protocols

both

subgroups

performed

equally

well

In

addition

the

inter-group

variability

in

detecting

positive

responses

was

reduced

in

phase

II

-LRB-

percentage

of

detected

responses

ranged

from

to

with

a

mean

of

-RRB-

as

compared

to

phase

I

-LRB-

percentage

of

detected

responses

ranged

from

to

with

a

mean

of

-RRB-

Experience

does

not

equal

performance

Among

the

centers

that

had

participated

in

phase

II

tetramer

stainings

had

been

performed

for

years

Similarly

the

experience

in

the

ELISPOT

technology

varied

between

and

years

For

both

techniques

we

could

not

find

any

correlation

between

the

years

of

experience

and

the

ability

to

detect

T-cell

responses

not

even

among

the

subgroups

of

moderate

or

low

T-cell

responses

-LRB-

not

shown

-RRB-

Discussion

Whenever

new

techniques

are

introduced

to

the

scientific

community

they

are

first

only

available

to

a

small

group

of

expert

laboratories

If

these

assays

are

robust

and

applicable

for

specific

research

or

routine

applications

they

spread

to

the

international

community

In

general

the

original

protocol

then

undergoes

several

adaptations

in

order

to

meet

specific

needs

On

the

one

hand

changes

can

be

beneficial

and

result

in

the

improvement

of

protocols

On

the

other

this

evolutionary

process

leads

to

employment

of

many

different

protocol

variants

limiting

comparison

of

the

study

results

obtained

by

different

laboratories

Thus

standardization

approaches

should

be

omitted

during

the

initial

development

but

are

absolutely

required

when

assays

have

become

firmly

established

In

recent

years

several

activities

aiming

at

the

harmonization

of

techniques

used

to

monitor

the

presence

of

antigen-specific

T-cells

have

been

initiated

for

ELISPOT

-LSB-

-RSB-

tetramer

staining

-LSB-

-RSB-

and

ICS

-LSB-

-RSB-

While

these

studies

showed

the

feasibility

general

applicability

and

the

diversity

of

performance

among

participants

they

were

not

designed

to

either

systematically

investigate

the

influence

of

distinct

protocol

variables

nor

to

test

whether

changes

to

these

parameters

can

lead

to

a

global

improvement

of

the

group

The

CIMT

monitoring

panel

is

the

first

initiative

that

has

now

introduced

the

two-step

approach

proposing

a

strategy

where

technical

variables

that

influence

the

performance

of

a

defined

assay

are

first

systematically

identified

-LRB-

first

step

-RRB-

followed

by

a

new

testing

phase

where

resultant

protocol

changes

are

validated

under

controlled

conditions

within

the

same

group

of

investigators

-LRB-

second

step

-RRB-

As

soon

as

a

number

of

protocol

variables

that

might

have

influenced

the

sensitivity

and

the

quality

of

the

tests

were

identified

in

the

first

phase

of

our

study

it

was

decided

to

validate

this

finding

in

a

second

phase

Because

this

two-step

approach

was

not

initially

foreseen

the

second

phase

was

performed

with

PBMC

samples

obtained

from

different

donors

than

those

used

in

the

first

round

The

distribution

and

the

frequencies

of

detectable

T-cell

responses

directed

against

the

chosen

model

antigens

were

different

in

the

first

and

second

group

of

donors

-LRB-

Fig.

-RRB-

precluding

a

direct

comparison

of

the

results

obtained

in

both

phases

of

the

panel

To

circumvent

this

problem

two

assay-specific

frequency

thresholds

were

introduced

that

allowed

us

to

distinguish

classes

of

T-cell

responses

-LRB-

low

moderate

and

high

-RRB-

-LRB-

Fig.

b

-RRB-

Clearly

high-frequency

T-cell

responses

were

detected

irrespective

of

the

protocol

used

and

as

such

did

not

allow

the

identification

of

factors

that

exert

a

strong

influence

on

the

sensitivity

and

variability

of

the

protocols

used

Relevant

parameters

could

only

be

detected

when

the

comparison

was

focused

on

the

detection

of

T-cells

that

are

present

at

low

to

moderate

frequencies

in

PBMC

This

finding

should

be

taken

into

account

when

selecting

model

antigens

for

use

in

monitoring

panels

-LSB-

-RSB-

in

particular

by

laboratories

that

are

interested

in

the

detection

of

peripheral

tumor-specific

T-cells

which

are

often

present

at

low

frequencies

even

after

vaccination

Although

our

experiments

do

not

specifically

address

the

question

of

detection

limits

for

the

ELISPOT

and

tetramer

assays

we

could

detect

a

high

variability

in

the

sensitivity

of

protocols

used

by

the

different

participants

The

majority

of

labs

-LRB-

y

-RRB-

is

able

to

detect

responses

with

a

frequency

above

per

T

cells

in

the

tetramer

assay

or

responses

with

a

frequency

above

per

PBMC

in

the

ELISPOT

Note

that

some

of

the

centers

could

reliably

detect

a

response

with

a

frequency

of

about

per

T

cells

in

the

tetramer

assay

and

about

per

PBMC

in

the

ELISPOT

assay

These

low

frequencies

are

in

the

range

of

that

is

commonly

reported

as

the

detection

limit

for

internally

validated

protocols

for

both

technologies

-LSB-

own

unpublished

observations

-RSB-

Another

important

task

of

standardization

efforts

should

be

to

decrease

the

variation

of

results

obtained

in

a

group

of

several

laboratories

down

to

the

stable

and

low

values

-LRB-

-RRB-

that

can

be

reproducibly

found

within

single

labs

In

order

to

quantify

the

variation

of

results

among

laboratories

we

calculated

the

coefficient

of

variation

for

all

reactivities

of

the

two

panel

phases

The

CVs

were

determined

on

the

base

of

centers

that

were

able

to

detect

the

respective

T

cell

response

and

the

results

are

shown

in

supplementary

Tables

b

As

expected

the

CVs

we

found

in

our

inter-laboratory

testing

project

were

higher

than

those

reported

from

intra-center

analysis

-LSB-

-RSB-

In

the

ELISPOT

assay

the

background

spot

numbers

obtained

by

the

different

participants

varied

greatly

but

we

were

unable

to

correlate

this

finding

to

a

distinct

variable

Since

the

spontaneous

cytokine

secretion

impacts

significantly

on

the

sensitivity

of

this

assay

factors

that

especially

influence

the

non-specific

spot

production

possibly

the

medium

type

or

serum

source

will

need

to

be

systematically

analyzed

in

a

separate

study

The

main

conclusions

from

our

study

have

been

drawn

on

the

basis

of

subgroup

analyses

Although

the

CIMT

panel

in

general

-LRB-

centers

in

this

initial

action

-RRB-

and

consequently

the

subgroups

formed

during

the

analysis

were

rather

small

we

could

already

identify

statistically

significant

differences

in

the

ability

to

detect

positive

responses

We

concluded

that

the

number

of

counted

T-cells

is

the

most

influential

crucial

factor

for

the

tetramer

assay

and

that

the

combination

of

a

resting-time

and

a

high

number

of

PBMC

leads

to

increased

sensitivity

in

the

ELISPOT

assay

This

suggests

that

the

impact

of

the

identified

technical

variables

on

the

quality

of

the

assays

is

high

In

order

to

identify

those

protocol

variables

that

lead

to

more

subtle

differences

a

larger

group

of

participants

would

be

needed

In

addition

to

the

systematic

identification

of

variables

that

correlate

with

sensitivity/insensitivity

of

various

assays

inter-laboratory

testing

projects

also

allow

the

rapid

evaluation

of

individual

performance

among

a

group

Interestingly

the

finding

that

experienced

laboratories

did

not

perform

better

than

laboratories

which

recently

applied

these

techniques

strongly

suggests

that

non-optimal

protocols

once

established

in

a

lab

can

commonly

be

maintained

for

several

years

Periodic

comparison

of

local

protocols

with

those

of

other

centers

is

recommended

Even

if

a

new

staff

member

uses

an

established

protocol

it

is

recommended

to

have

them

participate

in

inter-laboratory

testing/teaching

exercise

Regular

participation

in

multi-center

comparisons

could

thereby

help

to

optimize

and

validate

participants

performance

over

time

and

to

maintain

sensitive

protocols

or

minimal

standards

This

is

of

great

importance

when

material

from

expensive

clinical

trials

has

to

be

analyzed

All

data

from

the

CMV-serology

from

the

pre-testing

experiments

and

from

the

results

generated

by

the

participating

laboratories

in

ELISPOT

and

tetramer

staining

were

taken

together

for

each

donor

in

order

to

qualitatively

validate

the

presence

of

CMV

and

influenza-specific

T-cells

To

estimate

the

quantity

i.e.

the

frequency

of

specific

T-cells

in

each

donor

we

calculated

the

average

of

all

qualitatively

positive

results

as

well

as

the

standard

deviations

This

procedure

constitutes

only

an

approximation

of

the

real

number

of

antigen-specific

cells

present

in

a

given

sample

and

can

not

be

taken

as

a

method

for

determining

absolute

T-cell

frequencies

Cell

samples

that

contain

pre-defined

numbers

of

antigen-specific

T-cells

-LRB-

e.g.

spiked

T-cell

clones

-RRB-

especially

tumor-reactive

T-cells

are

not

easily

available

for

use

in

multi-center

comparisons

although

such

standard

samples

are

urgently

needed

We

see

this

as

one

major

bottle-neck

for

the

optimization

and

standardization

of

immunomonitoring

techniques

Methods

to

generate

such

standard

samples

for

broader

use

will

therefore

be

elucidated

with

high

priority

in

the

near

future

for

the

next

phases

of

this

international

collaboration

Another

big

challenge

will

be

to

define

accepted

rules

for

the

settings

of

the

equipment

used

in

these

analyses

-LRB-

flow

cytometer

or

ELISPOT

reader

-RRB-

in

order

to

uniformly

process

and

analyze

the

raw

data

Ten

from

eleven

laboratories

that

performed

the

ELISPOT

assay

in

the

first

phase

used

an

ELISPOT

reader

for

spot

counting

It

is

known

that

spot

counts

between

centers

can

differ

significantly

and

this

may

be

explained

by

the

use

of

different

reading

machines

different

settings

for

the

same

type

of

machine

or

by

the

experience

of

the

operator

Within

this

group

four

different

commercially

available

reading

systems

were

used

-LRB-

supplementary

Table

-RRB-

We

were

not

able

to

identify

differences

between

the

types

of

ELISPOT

readers

A

new

ELISPOT

panel

phase

is

currently

in

preparation

that

will

specifically

focus

on

the

performances

of

different

ELISPOT

readers

and

try

to

introduce

tools

to

control

inter

center

variation

In

addition

of

the

participant

reported

on

the

use

of

live/dead

cell

discrimination

on

thawed

PBMC

samples

for

the

FACS-based

experiments

Whether

the

combination

of

staining

with

Ab/HLA-tetramers

and

vital

dyes

or

with

a

resting

phase

is

beneficial

for

increasing

the

sensitivity

of

the

tetramer

staining

assay

could

be

addressed

in

future

testing

actions

Results

from

a

proficiency

panel

of

laboratories

from

nine

different

countries

in

which

the

ELISPOT

assay

was

validated

are

now

also

being

reported

-LSB-

-RSB-

This

initiative

conducted

under

the

aegis

of

the

Cancer

Vaccine

Consortium

-LRB-

CVC

-RRB-

was

mainly

designed

to

offer

an

external

validation

to

the

participating

laboratories

but

the

in

depth

analysis

of

the

obtained

data

sets

lead

to

similar

findings

and

recommendations

as

the

CIMT

monitoring

panel

It

confirmed

that

a

resting

phase

of

cells

prior

to

addition

to

the

ELISPOT

plates

is

advantageous

and

should

therefore

be

generally

recommended

Furthermore

a

long

year

experience

in

a

technology

did

not

guarantee

for

a

sensitive

test

and

failure

to

detect

specific

T

cell

responses

concentrated

on

the

weak

responses

The

fact

that

two

independent

initiatives

come

to

similar

findings

is

surely

notable

and

shows

the

necessity

to

carry

on

running

proficiency

panels

Last

but

not

least

we

would

like

to

stress

that

even

the

best

guidelines

and

protocols

alone

can

not

guarantee

good

performance

Monitoring

of

antigen-specific

T-cell

responses

requires

skills

as

well

as

experience

Participation

in

immunomonitoring

panels

can

not

compensate

for

the

need

to

constantly

educate

and

train

staff

and

to

develop

specific

expertise

for

covering

individual

needs

Nevertheless

we

strongly

believe

that

by

organizing

further

two-step

inter-laboratory

testing

projects

the

CIMT

monitoring

panel

will

be

able

to

improve

the

sensitivity

of

the

assays

used

for

immunomonitoring

as

well

as

to

actively

participate

in

the

harmonization

of

these

assays

which

is

required

to

enable

the

comparison

of

immunotherapeutic

trials

performed

in

different

centers

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

ESM

-LRB-

PPT

kB

-RRB-

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

elispot
tetramer
standardization
interlaboratory testing