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The radioallergosorbent test in the in vitro diagnosis of multiple reaginic allergy

A comparison

of

diagnostic

approaches

K. Aas,

M.D.,

and

S.

G.

0.

Johansson,

M.D.

Oslo, Norway,

The

clinical

resdts

of

allergy

and Uppsala,

Sweden

the

radioallergosorbent

diagnosis

by means

of

test

(RAST)

case history,

were

compared

and

with

skin

tests,

provocation

those

of

tests.

The

diagnostic

acmmy

of

RAST

 

as well

as

of

ease history

and skin

tests

depended

on

the

type

of

allergen.

The

ol;er-all

reliability

of

RAST

um

73

per

cent.

The

in

vitro

test

was

found

to

be

a

very

valuable

aid

as a screening

method

before

provocation

tests.

The

use

of

RAST

as a supplement

to

a carefully

collected

cnse

history

and

correctly

performed

and

critically

evaluated

skin

tests

made provocation

tests

superfluous

in

86

per

cent

of

the

patients

in

the

present

study.

 

The

radioallergosorbent

 

test

(RAST),

developed

for

the

detection

in

serum of reaginic

(IgE

)

antibodies

against

particular

allergens,1

has

been

reported

to be useful

for

the

in

vitro

diagnosis

of allergy.1-3

The present

study

was designed

to investigate

to what

extent

R.AST

may

replace

diagnostic

met,hods currently

in

use.

MATERIAL

AND

METHODS

Twenty-nine

allergy

to more

nontreated

than

patients

one substance,

were

selected

resulting

in

because

bronchial

they

suffered

from

asthma

and other

or supplement

immediate

symptoms.

typo

All

of

them

had

been

through

a

complete

allergy

investigation

 

thorough

and

repeated

cast

history

interviewing,

skin

tests,

and

provocation

comprising tests% 5 with

the

allergens

10 be tnstr,tl

by RAST

and with

other

allergens

as well.

 
 

Skin

tests

were

performed

 

as

scratch

and

prick

tests,

and,

if

these

were

negative.

intradermal

tests

also

were

carried

out

by generally

accepted

methods.6

The extracts

used were

obtained

from

Allergologisk

Laboratorium,

Copenhagen,

Denmark

(ALR),

and

Nyegnnrd

and

Co. A/S

(Nyco),

Oslo, Norway

(Table

I).

Dry

pollen

was obtained

from

Cernelle

AH,

Rwedrn.

Tho

same

extracts

were

used

in

provocative

tests

as

in

skin

testing.

Stock

solutions

n’ere

diluted

in

0.9 per

cent

saline

containing

 

0.5 per

cent

phenol.

The

latter

solution

was used al$o

as

a

control

in

addition

to

the

control

solutions

supplied

commercially

with

the

allergen

extracts.

The

highest

concentrations

of

each

allergen

extract

used

in

skin

testing

and

in

provocative

tests

were

as shown

in

Table

I.

As

negative

(-j

Controls,

the

control

solutions

supplied

by

the

extract

manufacturers

mere used.

Histamine

solut,ions

of

0.1 mg.

per

milliliter

concentration

were

used

as positive

controls

for

intrxcutnneous

tests,

rind

histamine

solutions

Prom

the

Pediatric

Department

and

Pediatric

Research

Institute,

Rikshospitalrt

Unirrrsity

Hospital,

Oslo,

and the Blood

Centre,

University

Hospital,

Uppsala.

Supported

by

the

Swedish

Medical

Research

Council

Giant

‘No. k71-16S-1050iC.

 

Received

for

publication

 

Feb.

26, 1971.

 

Reprint

requests

to:

Ii.

Aas,

Pediatric

Research

Institute,

Allergy

TJnit,

Rikshospitnlet

 

Uni-

versity

Hospital,

University

of Oslo,

Oslo I, Norway.

 

Vol.

48, x0.

3, pp. 234-148

 

VOLUME

NUMBER 3

48

Radioaliergosorbent

test

135

TABLE

I. Type

of

material

used

in

the

clinical

tests

 

Material

Highest

concentration

used

(loll

extract

(ALK)

 

Scratch

tests,

20 mg.%

of total

protein

corresponding

to approx.

l/2,000

w/v

House

dust

extract

(ALK)

 

Intradermal

tests,

l/10,000;

provocation

 
 

tests,

l/100

w/v

 

House

dust

extract

(Nyco)

 

Scratch

tests,

l/100;

 

if

negative,

intradermal,

 
 

l/1,000;

provocation

tests,

l/20

w/v

 

Birch

and

timothy

pollen

extract

(Nyco)

Scratch

and prick

tests,

l/100

;

if

negative

 

intradermal,

1/10,000;

provocation

tests,

 

l/100

w/v

Rirch

and

timothy

pollen

grain

For

rllinoconjurlct,ival

 

provocation

 

tests,

pine

 

pollen

used as control

allergen

 

Horse

dander

extract

(Nyco)

 

Prick

test,

l/100;

if

negative,

intradermal,

 
 

l/10,000;

provoca.tion

tests,

l/100

w/v

of

1

mg.

per

milliliter

concentration

were

used

in

scratch

and

prick

tests

to

gnuge

the

individual

 

skin

reactivity

of

each patient.

Skin

tests

were

read

and recorded

after

PO minutes,

only

urticarial

wheal

reactions

being

considered.

The

reaction

to

the

histamine

test

was

re-

corded

as three

plus

(3t),

and

test

reactions

were

compared

with

the

histamine

control

and

the

negative

control.

Reactions

smaller

than

that

of

the

histamine

test

were

recorded

as one

(l+)

or two

plus

(et),

and reactions

larger

than

that

produced

by histamine

received

atlditional

plus

marks,

such that

each new plus

indicated

a twofold

increase

in

the

wheal

area.

 

Each

patient

was

submitted

to provocation

tests

with

all

allergens

included

in

this

study

regardless

of

the

reaction

to

the

skin

test.

Provocation

tests

were

performed

as bronchial

inhalation

tests

with

nebulized

allergens

as described

in

detail

elsewhere,4

and,

if

these

were

negative,

increasing

concentrations

of

the

extract

were

applied

to

the

conjunctival

sac

or

nostrils.

If

the

latter

tests

were

negative

for

pollen

extracts,

dry

pollen

was

applied.

Controls

were

used

as described

before,4

including

allergens

that

the

patient.

obviously

should

tolerate

according

to

the

case

history.

The

degree

of

hypersensitivity

was

scored

according

to

the

lowest

concentration

of

allergen

needed

to

elicit

a positive

reaction

(Table

II).

The

presence

or

absence

of

allergy

to

fish

was

investigated

by

means

of

single-blind

test

meals

with

the

fish

concealed

in

hamburgers.5

Passive

transfer

tests

were

performed

as described

previously.7

For

comparison

of

the

accuracy

of

the

various

diagnostic

approaches,

a score

system

was

developed:

A

case history

suggesting

allergy

was

scored

from

0

to

+3.

Tf

the

history

did

not

give

any

evidence

whatsoever

as

to

whether

or

not

allergy

was

present,

0 was

given.

If

the

history

was

completely

convincing

about

allergic

reactions

to

a

given

substance,

t3

was

assigned,

and

other

case histories

were

scored

accordingly.

Similarly,

a score from

0 to

-3

was

given

for

histories

indicating

to different

degrees

that

the

patient

 

could

not

be allergic

to

the

particular

allergen,

minus

three

meaning

that

the

patient

obviously

 

tolerated

the

particular

allergen.

Reactions

to

skin

tests

were

recorded

from

0

to

+4.

For

this

the

results

of

scratch

tests

were

considered

for

the

fish

extract,5

prick

test

results

for

the

horse

dander

and

the

pollen

extracts,

and

the

results

of

intradermal

tests

were

considered

in

the

scoring

system

when

house

dust

extracts

were

employed.

As

indicated

in

Table

III,

they

were

scored

as

0

t,o

t4

if

correlated

 

with

a clinica

history

of

allergy

and

from

-4

to

0

if

not

correlated

with

clinical

allergy.

 
 

The

radioallergosorbent

test

is

an

antiglobulin

reaction

based

upon

the

detection

 

of

allergen

antibodies

of

IgE

type,

by

IzsI-labeled

anti-IgE,

on particles

to which

allergens

have

been

coupled.1.

3

In

this

study

RAST

was

performed

using

the

same

batches

of

allergen

particle

complexes

(APC)

and

IzbI-labeled

anti-IgE

as

in

the

earlier

study.3

The

APC

were

prepared

in

the

following

way:

One milliliter

of

an a.llergen

extract

of

the highest

available

concentration

was

mixed

with

100 mg.

of

CNBr-activated

cellulose

particles

in

O.lM

carbonate

136

Aas

TABLE II

and

Johansson

Sensitivity

J. ALLERGY CLIN.

IMMUNOL.

SEPTEMBER 1971

 

Result

of

provocation

tests

 

SCOVS

Negative

 

0

65

(RASTTI)

52

0.1

Positive

with

pollen

grains

(rhinoconjunc-

 
 

tival

tests

only)

 

1

8

(RAST23)

 

2

5

Positive

on provocation

with

full

dose of

 
 

highest

concentration

(l/100

w/v)

2

20

(RAST,S)

10

3.;

Positive

on provocation

with

reduced

dose of

 
 

highest

concentration

(l/100

 

w/v)

3

9

(BAST,-3)

 

G

4.2

Positive

on provocation

with

dilution

of

 

allergen

l/I,000

w/v

4

33

(RASTs3)

10

4.2

Positive

on provocation

with

dilution

of

 

allergen

l/10,000

w/v

5

6

(RAST53)

 

5

5.2

Positive

on provocation

with

dilution

of

 

allergen

l/100,000

w/v

or less

 

6

13

(RASTs3)

 

8

4.2

TABLE III. Score system

for

the

results

of

skin

tests

correlated

with

presence

or

absence

of

clinical allergy

to

the

particular

allergen

(retrospectively)

 
 

Skin

test

reaction

 

Score

for

positive

allergies

Score

for

negctive

allergies

 

0

 

+

+1

1;

“+

+2

-2

3+

+3

-1

4+

or

more

 

VI

0

In

instances

of

a

clinical

history

without

any

information

concerned

with

the

allergen

in

question,

the

skin

test

results

were

scored

as

for

(‘positive

allergies.”

 

buffer,

pH

8.4. After

incubation

for

72 hours

at

25’

C., the

suspension

was washed

with

0.5M

carbonate

buffer

and

incubated

in

O.lM

ethanolamine-OJM

NaHCO,

overnight

at

25”

C.

Finally

the

product

was

washed

with

O.lM

acetate

buffer,

pH

4, followed

by

0.5M

carbonate

buffer,

pH

8.4. The

APC

were

stored

at

+4”

C.

in

O.lM

phosphate

buffer,

pH

7.4, containing

one per

cent

tween

210and

0.2 per

cent

human

serum

albumin.

 

A RAST

experiment

is performed

in the following

way:

Fifty

microliters

of reaginic

serum

is

incubated

at

room

temperature

overnight

with

approximately

500

pg

of

APC.

After

washing,

100

81 of

labeled

anti-IgE,

corresponding

to approximately

60,000

counts

per

minute

(c.p.m.)

is added

 

incubated

as above

and washed.

The

amount

of

radioactivity

bound

to

the

APC

and the mixture is measured

in

a scintillation

detector.

The

c.p.m.

value

obtained

is com-

pared

with

a standard

curve

consisting

of

serial

dilutions

of

a serum

with

a

high

titer

of

reagins

to

birch

allergen.

This

standard

serum

has been

considered

to

contain

1,000 arbitrary

units.

As

little

as

1

to

3 units

 

can

be distinguished

from

the

background

uptake

of

radio-

activity.

For

allergy

diagnosis

it

was possible

to simplify

the quantitative

aspects8

and express

the

RAST

results

on

an

8 score

scale:

less than

3 units

was

regarded

as a negative

reaction,

0;

3

to

5.6 units,

1;

5.7

to

9.9 units,

2;

10

to

29 units,

3;

30

to

99 units,

4;

100 to

299 units,

 

5;

300

to

999 units,

6;

and

more

than

1,000 units,

7. Details

are

given

elsewhere.9

 

Tho

sera

studied

were

all

collected

before

provocation

tests

and

hyposensitization

treat-

ment.

They

were

analyzed,

diluted

1:2,

against

all

five

APC.

 

When

positive

(more

than

3

units),

 

they

were

retested,

if

possible,

in

two

dilutions

in

order

to

ascertain

the

parallelism

VOLUME

NUMBER 3

48

Radioallergosorbent

test

137

with

the

standard

curve.

In

the

case of

a high

titer

of

blocking

antibodies,

such a parallelism

is

not

obtained.3

The

RAST

score

given

represents

the mean

of

the two

analyses.

 

The

standard

error

of

the method

is about

20 per

cent.

 
 

In

the

present

study

RAST

was

considered

to

be positive

at

a score

of

3

or

more

and

negative

at

a

score

of

one

or

less.

Diagnostic

evidence

was

postulated

to

bc

present

for

allergy

when

the

total

score

sum for

case history

plus

skin

tests

was

5

or

more

points,

and

it

was

postulated

to

be lacking

 

(no

allergy)

when

thr

sum was

two

points

or

less.

Diagnostic.

evidence

was

postulated

 

also

to

be present

when

the

score

for

case history

plus

skin

test

plus

BAST

totallcd

 

+7

or

more,

and

no

allergy

diagnosis

 

was

postulated

to

bc established

if

the

total

sum was plus

two

or less.

The

postulations

were

tested

against

the results

of

the provo~:~

tion

tests.

 

RESULTS

The clinical

22; birch

dust,

of these patients

allergies

pollen,

were distributed

as follows:

cod, 11;

horse, 22;

house

The sera

(67 per

7; and timothy

a RAST

pollen,

score of 3 or more in 53 instances

17 patients,

respectively.

reacted with

cent).

In

the

64 instances

of no clinical

allergy

to

the

five

allergens,

the sera

reacted

in

RAST

with

one point

or

less in

52 cases (81

per

cent)

(Table

IV).

When

3 was chosen as the lower

diagnostic

limit

for

allergy

and

+l

was chosen

as the

upper

diagnostic

limit

for

no allergy,

an over-all

correlation

between the

clinical

diagnosis

and

RAST

was established

in

73 per

cent

of

all

cases. The

best correlations

were found

between negative

RAST

and no allergy

and between

positive

R.AST

and

allergies

in

which

the

clinical

reaction

was provoked

by

rcklativcly

low

concentrations

of

the

particular

allergen

(Table

II).

The

cor-

relation

depended

on

the

type

of allergen,

varying

from

9,3 per

cent

for

fish

allergy

to

59 per

cent

for

allergy

to house dust

(Table

V)

Dependence

on type

of allergen

was also characteristic

for

the reliability

of the clinical

evaluation

and

that

of

RAST

alone

as well

as in

combination.

The information

obtained

by case history

diagnosis, respectively,

by the appropriate

of the same cases. For

and skin tests

in

was sufficient

cent of

to establish

a positive

or a negative as determined

in

93 per cent

diagnosis

100 per

the casts of fish allergy

provocation

test. R,AST alone was diagnostic

house dust, the chance of establishing

a correct

by case history

and skin

tests was not more than

34 per cent. The corresponding

chance for

RAST

alone

was 59 per

cent

(Fi,.w 1). With

the combination

of cast

history

plus

skin

tests

plus

RAST,

a rather

good correlation

was found.

All

patients

w&h a total

sum of 7 or more were shown

to be allergic

to the partieulal

allergen,

and

all

patients

with

a total

sum of

2 or

less were

shown

to t,olerate

the

allergen.

By

this

combinat.ion

of methods

a correct

allergy

diagnosis

was

established

in

82 per

cent

of

all

instances

tested with

the five allergens

(Tahlc

VI).

DISCUSSION

Preliminary

studies

have

indicated

that

RAST

may

be most

valuable

for

experimental

work

within

the field of allergy

and that

it

is useful

for

the

in

vitro

tliagnosis

of

allergy.1-3, lo In

a preliminary

study

of

31 adult

patients

with

extrinsic

asthma

and hay

fever,

96 per cent of the diagnostic

provocation

 

tests

agreed

with

the

results

of

RAST.l

Stenius

and Wide’

found

83 per

cent

COY-

relation

between RAST

and the results

of skin prick

tests with

extracts

of house

138

TABLE

Aas

IV.

and

Johansson

Correlation

between

RAST score

and

the

presence

or

J. ALLERGY CLIN.

IMMUNOL.

SEPTEMBER 1971

absence

of

allergy

proved

by

provocation

tests

 

RAST

score

No.

of

patients

 

Positive

allergy

 

6

7

5

16

4

15

3

15

2

6

i

a

 

Negative

allergy

 

6

1

5

0

4

1

R

5

i

5

1

5

0

47

TABLE

V.

Accuracy

of

case

history

plus

method

in clinical

allergy

Allergen

Fish

Horso

Timotlly

pollen

 

Birch

pollen

House

dust

Average

 

“Presence

or absence

of

tNumbers

in parentheses

Allergy*

+ (11)

t

(18)

+ (22)

-

-c

+

-

+(

-

+

-c

7)

(17)

(12)

7)

(22)

(22)

7)

clinical

indicate

allergy

numbers

$Per

cent

of

correct

diagnoses.

skin

testing

as compared

with

RAST as diagnostic

Accuracy

history

(%I

plus

100

100

95

100

I

I

10082 1

of

skin

lOO$

g7

89

case

test

 

57

100

3

89*

;A

I

34

82

proved

by

means

of

provocation

of patients.

Accuracy

tests.

I % 1 of

91

94

68

(jg

65

67

86 76

66

71

I

I

I

93:

73

I

5o85

3

59

73

RAST

dust mite (Dermatophagoides cdinae)

house dust asthma. Fagerberg

selected patients with positive case histories and positive bronchial provocation

tests to extracts

in

patients

a

with

a history

cent

suggesting

in

and

Wide lo found

97 per

correlation

of dog epithelium.

A more thorough

analysis

of the accuracy

of RAST

as a diagnostic

test was

performed

by Berg,

Bennich,

and Johansson. 3 The over-all

agreement

between

the provocation In the present

study of Berg and associates,3 approximately 2 per cent of the tests seemed to be

influenced

samples these disturbances

control. a new diagnostic method is to wha.t

tests and RAST

study

a high

the overall

titer

questions

was found

to

be

74 per

cent

for

9 allergens.

In

the

of the

correlation

for 5 allergens

was 73 per cent.

by dilution

by

of blocking

antibodies.

However,

could be kept under

asked concerning

One of the first

VOLUME 48 Radioallergosorbent test 139 NUMBER 3 A/ History + skintest Appropriate provocalion test :

VOLUME

48

Radioallergosorbent

test

139

NUMBER 3

A/ History

+ skintest

Appropriate

provocalion

test

:

100%

B1R.A.S.T.

+ skintest Appropriate provocalion test : 100% B1R.A.S.T. Birch pollen 69% Birch pollen 76% I
+ skintest Appropriate provocalion test : 100% B1R.A.S.T. Birch pollen 69% Birch pollen 76% I
+ skintest Appropriate provocalion test : 100% B1R.A.S.T. Birch pollen 69% Birch pollen 76% I
+ skintest Appropriate provocalion test : 100% B1R.A.S.T. Birch pollen 69% Birch pollen 76% I

Birch

pollen

69%

Birch

pollen

76%

I

House

dust

34%

House

dust

59%

76% I House dust 34% House dust 59% FIG. 1. Comparison of diagnostic reliability of
76% I House dust 34% House dust 59% FIG. 1. Comparison of diagnostic reliability of

FIG.

1. Comparison

of diagnostic

reliability

of history

plus

skin

test

(A) and

RAST (B). Results

of

appropriate

provocation

test

=

100

per

cent.

TABLE VI. Total

score

for

case

history

plus

skin

test

results

plus

RAST

 

Instances

of

positive

Instances

of

negative

Total

score

allergy

diagnosis*

allergy

diagnosis

+7 or more

55

0

+6