Cellular Allergen Stimulation Test (CAST) 2003, a revie · Cellular Allergen Stimulation Test...
21
J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273 © 2004 Esmon Publicidad Review Article Cellular Allergen Stimulation Test (CAST) 2003, a review A.L. de Weck, M.L. Sanz Department of Allergology and Clinical Immunology, Clínica Universitaria de Navarra. University of Navarra, Pamplona, Spain Summary. Specific diagnosis of immediate type allergies, such as rhinoconjunctivis, asthma, urticaria/angioedema and anaphylaxis, particularly when IgE-mediated, traditionally rests on prick and/or intradermal skin tests and, since about 30 years, on the determination of allergen specific IgEs. Some cellular tests, i.e. tests determining the reactivity of blood cells in vitro, particularly basophils, to allergens, have been available for many years. The determination of histamine release has been widely used in allergy pathophysiological research but its routine application in allergy diagnosis has been restricted to few groups. Basophil degranulation, as determined by microscopic examination, was promoted by some groups in the 1980’s but has been largely abandoned since around 10 years ago; an alternative cellular test, based on the determination of sulfidoleukotrienes (LTC4, LTD4, LTE4) produced by IL-3 primed basophils stimulated by allergens in vitro, has been proposed. This test became available commercially in 1993 under the name of CAST (Bühlmann Laboratories, Allschwil, Switzerland). The CAST assay has been used in allergy diagnosis in a variety of indications, such as inhalation allergies, allergies to insect venoms, foods, occupational allergens and various drugs. A large number of reports on CAST diagnostic value, however, have been anecdotal. A meta-analysis of validated and well controlled studies encompasses 37 studies, 1614 patients and 1145 controls. This should definitely establish the value of this diagnostic test, particularly in instances where other in vitro or in vivo diagnostic tests are not reliable, such as food or drug allergies, as well as in non-IgE- mediated immediate hypersensitivity reactions. However, a number of questions about the CAST diagnostic assay are still open or have not been systematically explored. This may explain, in addition to the practical limitations inherent to all allergy cellular tests, why CAST has not yet become a very widely used assay worldwide, having gained broad acceptance in some countries but not in others. Key words: Sulphidoleukotriens, CAST, allergy, in vitro diagnosis Some cellular tests, i.e. tests determining the reactivity of blood cells in vitro, particularly basophils, to allergens, have also been available for many years. The determination of histamine release has been widely used in allergy pathophysiological research but its routine application in allergy diagnosis has been restricted to few groups [1,2]. Basophil degranulation, as determined by microscopic examination, was promoted by some groups in the 1980’s [3,4] but has been largely abandoned since. Around 10 years ago, an alternative cellular test, based on the determination of sulfidoleukotrienes (LTC4, LTD4, LTE4) produced by IL-3 primed basophils stimulated by allergens in vitro, has been proposed by one of us [5-8]. This test became available commercially in 1993 under the name of CAST (Bühlmann Laboratories, Allschwil, Switzerland). While the majority of studies in the literature have been performed with this test, some have used other commercially available anti-sulfidoleukotrienes (sLT)
Transcript of Cellular Allergen Stimulation Test (CAST) 2003, a revie · Cellular Allergen Stimulation Test...
A.L. de Weck, et al.
J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273 © 2004 Esmon Publicidad
Review Article
Cellular Allergen Stimulation Test(CAST) 2003, a review
A.L. de Weck, M.L. Sanz
Department of Allergology and Clinical Immunology, Clínica Universitaria de Navarra. University of Navarra,Pamplona, Spain
Summary. Specific diagnosis of immediate type allergies, such as rhinoconjunctivis, asthma, urticaria/angioedemaand anaphylaxis, particularly when IgE-mediated, traditionally rests on prick and/or intradermal skin tests and,since about 30 years, on the determination of allergen specific IgEs.Some cellular tests, i.e. tests determining the reactivity of blood cells in vitro, particularly basophils, to allergens,have been available for many years. The determination of histamine release has been widely used in allergypathophysiological research but its routine application in allergy diagnosis has been restricted to few groups.Basophil degranulation, as determined by microscopic examination, was promoted by some groups in the 1980’sbut has been largely abandoned since around 10 years ago; an alternative cellular test, based on the determinationof sulfidoleukotrienes (LTC4, LTD4, LTE4) produced by IL-3 primed basophils stimulated by allergens in vitro,has been proposed. This test became available commercially in 1993 under the name of CAST (BühlmannLaboratories, Allschwil, Switzerland).The CAST assay has been used in allergy diagnosis in a variety of indications, such as inhalation allergies, allergiesto insect venoms, foods, occupational allergens and various drugs. A large number of reports on CAST diagnosticvalue, however, have been anecdotal. A meta-analysis of validated and well controlled studies encompasses 37studies, 1614 patients and 1145 controls. This should definitely establish the value of this diagnostic test, particularlyin instances where other in vitro or in vivo diagnostic tests are not reliable, such as food or drug allergies, as wellas in non-IgE- mediated immediate hypersensitivity reactions.However, a number of questions about the CAST diagnostic assay are still open or have not been systematicallyexplored. This may explain, in addition to the practical limitations inherent to all allergy cellular tests, why CASThas not yet become a very widely used assay worldwide, having gained broad acceptance in some countries butnot in others.
Key words: Sulphidoleukotriens, CAST, allergy, in vitro diagnosis
Some cellular tests, i.e. tests determining the reactivityof blood cells in vitro, particularly basophils, to allergens,have also been available for many years. Thedetermination of histamine release has been widely usedin allergy pathophysiological research but its routineapplication in allergy diagnosis has been restricted to fewgroups [1,2]. Basophil degranulation, as determined bymicroscopic examination, was promoted by some groupsin the 1980’s [3,4] but has been largely abandoned since.
Around 10 years ago, an alternative cellular test, basedon the determination of sulfidoleukotrienes (LTC4, LTD4,LTE4) produced by IL-3 primed basophils stimulated byallergens in vitro, has been proposed by one of us [5-8].This test became available commercially in 1993 underthe name of CAST (Bühlmann Laboratories, Allschwil,Switzerland). While the majority of studies in the literaturehave been performed with this test, some have used othercommercially available anti-sulfidoleukotrienes (sLT)
Cellular Allergen Stimulation Test (CAST) 2003, a review
© 2004 Esmon Publicidad J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273
254
antibodies, usually of more restricted specificity (e.g.anti-LTC4, Cayman Laboratories).
Despite the fact that, particularly in Europe, severalgroups have published over the years positive andclinically validated studies on the use of CAST in allergydiagnosis for various indications, particularly for insectvenoms and drugs, other reports have been definitelynegative [9]. This apparent controversy has beenreinforced by some incomplete overviews [10,11] andrecently by a position paper from a study group of theGerman Society for Allergology and ClinicalImmunology, which basically concludes that for mostindications, there are not enough studies to recommendthe CAST assay for routine allergy diagnosis [12].Indeed, only three studies on CAST results are quotedin that position paper, one of them negative [9,13,14]although at that time more than 20 thoroughly controlledand clinically validated studies had been published. Itmay be difficult for many to objectively assess theliterature, since only one review in German from 1997has up to now been published [15]. In addition, many ofthe earlier publications were anecdotal, reportingrelatively few cases. Many reports have appeared in otherlanguages than English and/or are not available asMedline quotations. We have therefore found it usefulto attempt a better informed assessment, and to presenthere the first comprehensive review of published CASTresults in various clinical diagnostic indications.
This review includes, on the one hand and for theclinical indications of inhalants, food, venoms,occupational and drugs allergies, only 35 publications(with a total 504 patients investigated) which weconsider “anecdotal”, i.e. based on relatively few cases(less than 10) and/or insufficiently controlled. On theother hand, for the same diagnostic indications we havereviewed 37 studies, with a total 1614 patients, whichwe consider clinically validated, the allergy diagnosishaving been firmly established by other diagnosticcriteria (history, skin tests, specific IgE determinationsand, when indicated, provocation tests). Such studieshave also included suitable control groups with asufficient number of individuals (10 or more), totalling1145 controls.
Recently, another cellular allergy diagnostic test, theflowcytometric basophil activation test (BAT or FAST)1
has been developed and used in a number of clinicaldiagnostic indications similar to those of CAST. Theseresults have been recently reviewed [16,17]. Since thecombination of flow cytometric basophil activation andsLT determinations yields in some indicationscomplementary and improved diagnostic results, thesewill also be briefly discussed below2 .
1 Commercially available under the name of FLOW-CAST (BühlmannLaboratories, Allschwil, Switzerland) or BASOTEST (Beckton-Dickinson).
2 Commercially available combined FAST and CAST under the name of CASTCombi (Bühlmann Laboratories, Allschwil, Switzerland).
Allergy to inhalant allergens
In the early studies about CAST, the focus was seton classical aeroallergens such as grass pollens andhouse dust mites, in order to assess the usefulness andreliability of the test in IgE-mediated allergy and itsposition relative to other diagnostic tests, such as skintests, determination of allergen-specific IgEs andhistamine release test. After the first anecdotal studies,four validated studies encompassing 386 patients and247 controls, particularly with grass pollen (Loliumperenne, timothy) and with house dust mites(Dermatophagoides pteronyssinus) have confirmed thehigh sensitivity and specificity of CAST in theseindications (Table 1). In particular, the CAST testappears to be more sensitive than histamine release[18,19]. Since in most instances the patient’s history,skin tests and determination of allergen-specific IgEsuffice to establish the diagnosis and to provideindication for immunotherapy, the CAST, as anadditional and expensive diagnostic test, is rarelyjustified and has accordingly not found its way intoroutine allergology practice.
The matter might be different, however, in allergydiagnosis of early infancy, where skin tests are moredifficult to perform and where determinations ofallergen-specific IgE are less reliable and correlate lessclosely with the clinics. CAST has been tested repeatedlyin infant and children populations [20,21] but only onestudy has compared its diagnostic efficiency incomparison to skin tests and to allergen-specific IgEdeterminations and has concluded that CAST was notmore sensitive for allergy diagnosis in infants [22].Nevertheless, in individual cases CAST has shown thatit may be positive to inhalation and food allergens ininfants developing atopy while skin tests and specificIgE determinations are still negative [15]. In view ofthe increasing prevalence of paediatric IgE-mediatedallergies and of the importance of early detection forsecondary prevention, additional studies would be mostdesirable.
Detection of atopy
Following the same line of thought, early detectionof atopy and of the genetically conditioned ability todevelop high IgE levels in response to environmental orfood allergens has been increasingly emphasized. Withthe exception of the determination of egg or milk-specific IgE at the age of 6 – 12 months [23], otherparameters such as the determination of total or specificIgE or of allergen-specific T lymphocytes reactivity incord blood have proven disappointing and of poorpredictive value for the development of atopy.
In this respect, one study, although interpreted byits authors in a rather negative way [20] points out to
A.L. de Weck, et al.
J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273 © 2004 Esmon Publicidad
g
Ref
No
Au
tho
rA
llerg
en
sP
ati
en
tsN
bR
esu
ltC
on
tro
lsN
bR
esu
lts
Po
sit
ivit
y c
rite
ria
Co
mm
en
ts
A. A
necd
ota
l stu
die
s
6D
e W
eck e
t al.,,1992
5 inhala
nt alle
rgens
Rhin
itis
/Asth
ma
55 p
os. , corr
with S
T 1
.0
5D
e W
eck e
t al,1992
5 in
hala
nt alle
rgens
Rhin
iti/asth
ma
24
corr
ST
0.7
6; corr
sIg
E: 0.6
6
7D
e W
eck e
t al, 1
993
5 in
hala
nt alle
rgens
Rhin
itis
/ A
sth
ma
38
corr
St 0.8
5, corr
sIg
E 0
.72
HD
M a
llerg
ics
18
corr
sLT
to H
DM
/Anti-I
gE
0.8
6
117
Cosachov e
t al.,
1995
GP
, R
agw
eed,
Alle
rgic
s6
corr
ST
; corr
sIg
E g
ood
Mould
sm
ean s
LT
561 p
g/m
l
113
Hip
ler,
1994
GP
Alle
rgic
s10
hig
h c
orr
sLT
/ S
Tin
tra, in
tera
ssay C
V <
10%
102
Sain
te-L
audy, 1995
inhala
nt alle
rgens
Alle
rgic
s7
5 S
IgE
/sLT
pos,(
520-6
10 p
g)
sLT
100 p
g/m
l >
Bckg
SE
sLT
> H
R
(Gp5,c
at,H
DM
)2 s
IgE
neg/s
LT
pos
103
Sirid
opoulo
s,1
995
HD
M,G
Palle
rgic
s11
low
SE
Health
y4
hig
h S
P200 p
g >
Bckg
18,1
9F
err
er
et al ., 1
996
GP
, H
DM
HD
M a
llerg
ics
23
sig
nific
ant sLT
in p
atients
GP
alle
rgic
s10
corr
sLT
/HR
0.4
4
132
Sain
te-L
audy, 1996
HD
M5
5/5
pos (
sLT
130-7
10 p
g/m
l)N
on s
ensitiz
ed
hig
h S
Phum
an b
aso s
ensitiz
ed b
y
HD
M
hum
an b
asophils
passiv
e s
eru
m tra
nsfe
r (h
eat la
bile
)
inhib
itio
n H
R b
ut not sLT
by h
igh d
ose A
g
134
Medra
la e
t al,
1997
GP
Alle
rgic
s23
20/2
3 (
SE
87%
) -m
ean:1
671 p
g/m
lsLT
200 p
g/m
l >
Bckg
B. V
alid
ate
d s
tud
ies
111
Medra
la e
t al,
1997
HD
MA
llerg
ics
23
21/2
3 (
SE
91%
)-m
ean:1
521 p
g/m
lH
ealth
y15
12 s
LT
,ST
neg (
SP
100%
)sLT
200 p
g/m
l >
Bckg
3 s
LT
,ST
pos (
< 3
00 p
g/m
l)
98
Ferr
er
et al, 1
996
HD
MA
llerg
ics
62
sLT
pos.-
mean 1
731 p
g/m
lA
topic
s
30
mean S
LT
291 p
g/m
lcorr
HR
0.8
3; corr
sIg
E 0
.4
(ST
pos)
(HD
M S
T n
eg)
corr
ST
0.4
9
coor
ST
: 0.4
2; corr
sIg
E 0
.40
Health
y12
mean s
LT
55 p
g/m
lno c
orr
with a
sth
ma s
everity
hig
h S
E
18
Ferr
er
et al, 1
996
GP
Alle
rgic
s54
corr
ST
0.5
0; m
ean s
LT
2123 p
g/m
lA
topic
s
38
mean s
LT
940 p
g/m
lcorr
HR
0.6
7, corr
sIg
E/S
T 0
.5
(ST
pos)
(GP
ST
neg)
hig
her
sLT
in p
olle
n s
eason
Health
y9
mean s
LT
62 p
g/m
lsLT
T <
by a
ge >
40 y
rs
135
Rossi et al.,1
998
HD
M D
pt, D
fR
hin
iis/A
sth
ma
247
Dpt S
E 7
1%
,mean s
LT
969/p
g/m
lH
ealth
y137
Dpt S
P 8
7 %
,mean S
LT
240 p
g/m
ldefined b
y R
OC
curv
e
(ST
pos)
Df S
E 6
4%
-mean S
LT
1123 p
g/m
l(
ST
, sIg
E n
eg)
Df S
P 7
1 %
, m
ean s
LT
249 p
g/m
lD
pt: 4
70 p
g D
f: 5
23 p
g
the potential use of CAST as a screening test incord blood. Among 13 children with familyhistory of atopy, all were positive in CAST to amixture of classical paediatric allergens ( TOPCAST mixture of 21 allergens), while 24 out of91 children with negative family history werealso CAST positive. The authors concluded topoor specificity, but in view of the fact that about50% of the infants ultimately developing atopyhave no direct family history and that the childrenin that study were not followed up, a negativeconclusion is not yet justified. This study showsthat evidence of sensitisation is present alreadyat the time of birth and basophils may releasemediators in an allergen-specific way. FurtherCAST studies of newborns, if possible combinedwith flowcytometric determination of basophilactivation and clinical follow up would be verydesirable.
Another potential use of CAST for thedetection of atopy has been as a screening testin children and adults, mostly using a mixtureof allergens (TOP-CAST).
Among several such studies (Table 2), threevalidated ones encompassing 192 patients[24,25,26] have shown that CAST is indeedvery reliable as a screening test for IgE-relatedatopy, to the same or even higher degree thanscreening for multiple allergen-specific IgEs(e.g. Phadiatop) [24,25,27] but whether thisholds in infants has been questioned [22].CAST could theoretically be used for massblood screening, with greater reliability andlogistical simplification than skin tests [28],particularly if it were automated.
Food allergy
Although IgE-mediated food allergiesrepresent an important proportion of allergicdiseases, their diagnosis is markedly moredifficult than that of allergies to aeroallergens.Food allergies may take several forms, fromimmediate-type symptoms, such as anaphylaxis,urticaria, angioedema, asthma or rhinitis, to moredelayed manifestations often including gastro-intestinal symptoms. Beyond historyelaboration, which may be imprecise anddifficult, skin tests and determination of specificIgEs are often considered relatively unreliable[29]. For some foods (e.g. celery, hazelnut,carrot), the sharing of epitopes with some pollens(e.g. birch) often make the determination ofspecific IgEs meaningless, since immunologicalcross-reactivity is not associated with clinicalcross-reactivity [30].
Tabl
e 1.
Inh
alan
t all
erge
ns
255
Cellular Allergen Stimulation Test (CAST) 2003, a review
© 2004 Esmon Publicidad J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273
ab
eete
ct
oo
ato
py
Ref
No
Au
tho
rA
llerg
en
sP
ati
en
tsN
bR
esu
ltC
on
tro
lsN
bR
esu
lts
Po
sit
ivit
y c
rite
ria
Co
mm
en
ts
24
,27
De
rer,
19
95
TC
mix
HD
M/G
P a
llerg
ics
52
SE
86
%S
P 9
1 %
co
rr P
ha
dia
top
0.8
8
11
9,1
4S
hic
hijo
,19
95
HD
M,a
-Ig
EH
DM
Ato
pic
s1
3sL
T >
to
HD
M,
a-I
gE
He
alth
y5
IL3
do
es n
ot
incre
ase
sL
T >
in
pa
tie
nts
with
HR
5
0%
sL
T f
rom
no
rma
l d
on
ors
co
rr H
R +
IL3
0.8
3;
-IL
3 o
.58
12
1Z
ub
erb
ier,
19
96
a-I
gE
ch
ron
ic u
rtic
ari
a8
me
an
sL
T 5
86
pg
/ml
He
alth
y9
me
an
sL
T 5
94
pg
/ml
HR
< in
pa
tie
nts
IL-3
(2
0 n
g/m
l >
sL
T t
o a
-Ig
E
25
Me
dra
la,1
99
7T
C m
ixH
DM
/GP
alle
rgic
s2
42
4/2
4 (
SE
10
0 %
)H
ea
lth
y1
64
sL
T,
ST
po
s,
sL
T,
ST
po
s c
on
tro
ls <
10
00
pg
/m
at
Ag
10
0 n
g/m
l1
1 S
LT
, S
T n
eg
sL
T 2
00
pg
/ml >
Bckg
alle
rgic
s <
20
00
pg
/ml
1 s
LT
ne
g,
ST
po
s
26
Fe
rre
r, S
an
z 1
99
8H
DM
Dp
tR
hin
itis
/asth
ma
62
sL
T t
o a
-Ig
E ~
20
00
pg
/ml
He
alth
y
12
sL
T a
-Ig
E ~
70
0 p
g/m
l1
4.5
% n
on
re
sp
on
de
rs t
o a
-Ig
E
GP
Lo
lpR
hin
itis
/asth
ma
54
co
rr s
LT
a-I
gE
/Ag
0.8
IL-3
en
ha
nce
s s
LT
> H
R
20
Sze
pfa
lusi ,
19
99
TC
mix
Alle
rgic
ch
ildre
n5
64
5/5
6 p
os t
o a
-Ig
E
Me
an
sL
T t
o T
C:
10
49
pg
/ml
sL
T 2
00
pg
/ml >
Bckg
Co
rd b
loo
d,
fam
ily p
os
13
13
sL
T p
os:
to T
C:
41
9 p
g/m
lsL
T t
o a
-Ig
E c
orr
to
Ig
E le
ve
l;
Co
rd b
loo
d,
fam
ily n
eg
91
24
sL
T p
os.
sL
T t
o T
C d
oe
s n
ot
22
Tri
do
n ,
19
99
TC
mix
?A
sth
ma
tics 1
-3 y
rs o
ld4
4sL
T S
E:
22
%(6
/27
)h
igh
SP
sL
T 2
00
pg
/ml >
Bckg
sL
T p
os o
nly
wh
en
ST
or
(17
with
ou
t a
top
y y
et)
sIg
E S
E 5
2%
: S
T S
E 2
9%
sIg
E p
os
Tabl
e 2.
Det
ecti
on o
f at
opy
Tabl
e 3.
Foo
d al
lerg
ens
g
Ref
No
Au
tho
rA
llerg
en
sP
ati
en
tsN
bR
esu
ltC
on
tro
lsN
bR
esu
lts
Po
sit
ivit
y c
rite
ria
Co
mm
en
ts
A. A
necd
ota
l stu
die
s
11
3H
iple
r,1
99
5e
gg
,lo
bste
ra
llerg
ic4
4 s
LT
,ST
,sIg
E p
os
He
alth
y1
sL
T,
ST
, sIg
E n
eg
13
7S
tein
ma
nn
, 1
99
7e
gg
A
D c
hild
ren
17
10
Pro
v p
ow
s,
sL
T,S
T,s
IgE
po
sp
os D
BP
CF
C
4 P
rov p
os ,
sL
T,S
T,s
IgE
ne
g
3 P
rov n
eg
, sL
T,S
T,
sIg
E n
eg
10
6V
ila e
t a
l, 1
99
8ch
icke
n m
ea
tO
AS
1sL
T,
sIg
E,H
R,S
T p
os
34
Dia
z-P
era
les,
20
03
Pe
ach
,rP
ru p
3P
ea
ch
alle
rgic
s1
09
/1
0 p
os (
SE
90
%)
He
alth
y5
sL
T S
P 1
00
%S
LT
> H
R (
SE
50
%)
(ST
,sIg
E p
os)
rec P
ru p
3 =
na
tura
l P
ru p
3
B.
Va
lid
ate
d s
tud
ies
36
Mo
ne
ret-
Va
utr
in ,
19
99
va
rio
us f
oo
ds
urt
icri
a,a
sth
ma
27
22
/34
sL
T p
os (
SE
85
%)
He
alth
y2
41
/35
sL
T p
os (
SP
10
0 %
)sL
T 1
00
pg
/ml >
Bckg
sL
T O
K 2
4 h
rs a
fte
r sa
mp
ling
an
ap
hyla
xis
18
/31
BA
T p
os,
sIg
E S
E 9
1%
1/3
4 B
AT
po
s(
SP
10
0%
)B
ckg
< 1
00
pg
/ml
hig
h s
po
nta
ne
ou
s H
R (
7/1
1)
pa
ssiv
e s
eru
m t
ran
sfe
r w
he
n
sIg
E <
35
kU
> 3
.5 k
U
33
Vila
et
al, 1
99
9va
rio
us f
oo
ds
urt
ica
ria
94
0sL
T S
E 8
1%
,sIg
E S
E 7
7 %
He
alth
y2
0sL
T (
SP
98
%)
cu
toff
fro
m R
OC
cu
rve
ssL
T
po
s in
an
ap
hyla
xis
an
ap
hyla
xis
15
HR
SE
84
.5 %
, 3
3 P
rov p
os
me
an
sL
T a
-Ig
E 1
05
9 p
g/m
lsL
T 1
10
pg
/ml n
on
ato
pic
sco
rr S
T 0
.50
; co
rr s
IgE
0.3
5
rhin
itis
5m
ea
n s
LT
a-I
gE
13
27
pg
/ml
me
an
sL
T A
g 9
5 p
g/m
lsL
T 2
50
pg
/ml a
top
ics
co
rr H
R 0
.67
OA
S 1
3m
ea
n s
LT
Ag
83
6 p
g/m
lA
top
ics
15
sIg
E (
SP
95
%)
7 a
top
ic p
atie
nts
po
s s
LT
,ST
exe
rcis
e 5
no
fo
od
alle
rgy
me
an
sL
T a
-Ig
E 1
63
0 p
g/m
lH
R,
sIg
E b
ut
ne
g D
BP
CF
C P
rov !
ga
str
oin
test
3m
ea
n s
LT
Ag
23
1 p
g/m
l
30
Ba
llme
r,2
00
3h
aze
lnu
t,O
AS
Pro
v p
os
43
sL
T S
E 7
0%
(10
ug
Ag
)H
ea
lth
y2
0sL
T S
P 1
00
%sIg
E S
E 6
6%
ce
lery
,ca
rro
tB
irch
alle
rgic
ssL
T S
E 8
4%
(1
00
ug
Ag
)
sT
.sIg
E p
os
41
sL
T S
E 1
2 %
(1
0u
g A
g)
Bir
ch
alle
rgic
es
42
sL
T S
P 8
8 %
(1
0u
g A
g)
Pro
v n
eg
sL
T S
E 3
2%
(1
00
ug
Ag
)P
rov n
eg
sL
T S
P 6
8%
(1
00
ug
Ag
)
256
A.L. de Weck, et al.
J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273 © 2004 Esmon Publicidad
257
Tabl
e 4.
Ins
ect V
enom
s.
Ref
No A
uth
or
Allerg
en
sP
ati
en
tsN
bR
esu
ltC
on
tro
lsN
bR
esu
lts
Po
sit
ivit
y c
rite
ria
Co
mm
en
ts
A. A
neccd
ota
l stu
die
s
108
Maly
, 1994
bee,w
asp
bee/w
asp a
llerg
ics
15
corr
ST
0.6
; corr
sIg
E 0
.46
Healthy
10
SP
94 %
>
mean c
ontr
ols
+ 2
SD
sLT
and H
R d
issocia
ted
corr
HR
0.3
6 -
SE
87 %
103
Sirid
opoulo
s,1
995
bee,w
asp
chid
ren
5 5
/5 s
LT
pos
Healthy
6nr
113
Hip
ple
r, 1
995
bee,w
asp
bee/w
asp a
llerg
ics
14
alm
ost com
ple
te c
orr
with
nr
his
tory
, sIg
E
107
Sabbah , 1
997
mosquito
anaphyla
xis
1sLT
(637 p
g)
, B
AT
, S
IgE
pos
sLT
24 p
g/m
l
109
Pere
ira-S
anto
s , 2
002
bee,w
asp
bee a
llerg
ics
14
sLT
SE
:93%
; S
T S
E 7
1%
ato
pic
5 >
200 p
g/m
lC
AS
T p
os in S
T, sIg
E n
eg
sIg
E S
E 7
1%
40,4
Eberlein
-König
, 2
003
bee.w
asp
bee/w
asp a
llerg
ics
14
wasp s
LT
SE
86 %
Healthy
5all
sLT
, sIg
E n
eg
sLT
> 6
81 p
g/m
l (b
ee)
6/2
8 s
T n
eg
, B
AT
an
d C
AS
T p
os
be
e s
LT
SE
50
%sIg
E n
eg
sL
T >
86
4 p
g/m
l (w
asp
)sL
T c
orr
BA
T 0
.79
B. C
on
tro
lled
stu
die
s
112
Höxte
rmann,1
995
wasp
wasp a
llerg
ics
25
sLT
SE
92 %
Healthy
10
sLT
SE
90%
> m
ean c
ontr
ols
+ 2
SD
corr
sLT
/sIg
E 0
.94
corr
ST
0.8
8;c
orr
sIg
E 0
.84
sIg
E n
eg
co
rr s
LT
/ST
0.8
8
sLT
bee S
E 1
00%
; w
asp S
E 8
3%
37,1
Maly
, 1
996,1
997
bee, w
asp
bee/w
asp a
llerg
ics
23
17 S
T p
os S
LT
88 %
HR
59%
Healthy
10
sLT
SP
90 %
> m
ean c
ontr
ols
+ 2
SD
dis
socia
tion s
LT
and H
R
6 S
T n
eg
,
SL
T 6
6%
bee S
P 7
7%
; w
asp S
P 1
00%
co
rr S
T :
sL
T >
HR
corr
ST
0.6
; corr
sIg
E o
.46
13
Cahen , 1
997
bee 3
1bee/w
asp a
llerg
ics
66
bee S
E 7
3%
; w
asp S
E 6
8%
healthy
13
bee S
P 7
1%
> m
ean c
ontr
ol +
2 o
r 3 S
DS
E n
ot better
w 3
Ag d
oses
wasp 2
7sIg
E n
eg
wasp S
P 1
00%
19 %
a-I
gE
neg,
?? 8
138
Sabbah, 1998
bee 8
local re
action
12
sLT
SE
87%
, B
AT
SE
100%
healthy
10
sLT
SP
100%
sLT
250 p
g/m
l >
bckg
SLT
and B
AT
best coore
late
d
wasp 4
0syte
mic
reaction
23
ST
SE
75%
, S
IgE
SE
86%
sIg
E n
eg
to c
linic
al his
tory
horn
et 1
ana s
hock
9H
R S
E 9
1%
100
Hip
ler,
1999
bee,w
asp
bee/w
asp a
llerg
ics
84
bee s
LT
SE
93%
: sIg
E S
E 8
7%
healthy
10
bee s
LT
SP
67%
; sIg
E S
P 7
0%
nr
hig
h r
epord
ucib
ility
local re
actions
6w
asp s
LT
SE
96%
; sIg
E S
E 9
2%
sIg
E n
eg
wasp s
LT
SP
68%
; sIG
E S
P 5
7%
sLT
not corr
with s
everity
84
Szym
anski, 1
999
bee 1
9alle
rgic
ST
,sIg
E p
os
43
sLT
maen ~
5000 b
efo
re IT
sLT
>600 in 1
0/1
1 p
atients
wasp 1
8pseudo S
T,s
IgE
neg
11
sLT
mean ~
1500 a
fter
ITpseudoalle
rgic
(S
T,s
IgE
neg)
VIT
failu
re7
42
Mart
i, 1
999
Culic
oid
es
hors
es w
ith s
um
mer
12
10 s
LT
pos (
SE
83 %
)healthy
16
sLT
SP
100 %
> m
ean c
ontr
ols
+ 3
SD
corr
sLT
/ H
R 0
.95
mosquito
eczem
a(3
59 p
g/m
l)
105
Sain
teLaudy, 2002
bee,w
asp
local re
action
12
sLT
SE
100%
; B
AT
SE
100%
stu
ng w
/o10
sLT
SP
100%
>
160 p
g/m
l
horn
et
syste
mic
reactions
25
ST
SE
85%
; H
R S
E 8
9 %
reaction
a-I
gE
> 3
00 p
g/m
l
ana s
hock
9
38
Anlik
er,
2003
bee 3
5syste
mic
reactions
96
sLT
SE
92%
; sIg
E S
E 7
0%
contr
ols
fro
m r
efs
58,5
9 3
SD
> c
ontr
ols
means
23 S
T a
nd s
IgE
neg :
20
wasp 6
1S
T S
E 7
6%
CA
ST
pos (
87%
)
99
Bore
lli,2
003
bee 2
045
sta
tus a
fter
3 y
rs IT
, te
st at 6,1
2,
>292 p
g 0
.01 u
g b
ee
no v
alu
e in m
onitoring IT
wasp 2
3
24,3
6 m
o, In
23, rise in S
T tre
shold
> 2
39 p
g 0
.01 u
g w
asp
16 fie
ld s
ting n
eg, all
still
CA
ST
pos
severa
l positiv
e c
ontr
ols
ST
pos
11
5
S
ch
ere
r, 2
00
5
be
e 3
5
an
ap
hyla
xis
14
3
1
50
*
No
n
40
**
*
**
**
**
wa
sp
72
loca
l re
act
7
se
nsitiz
ed
b
ee
+w
asp
43
*
b
ee
: S
E S
LT
92
%,
sIg
E,
97
%,
sT
97
%,
BA
T 9
3%
W
asp
: S
E s
LT
90
%,
sIg
E 8
8%
, sT
97
%,
BA
T 8
5%
**
be
e S
P s
LT
93
%,
sIg
E 9
2%
, S
T 9
7%
, B
AT
94
%
Wa
sp
SP
sLT
95
%,
sIg
E 9
3%
, S
T 9
0%
, B
AT
95
%**
* c
uto
ffm
ea
ns +
3 S
D a
nd
10
0 p
g >
BcK
g,
S1
> 2
****
Be
tte
r d
iscri
min
atio
n b
etw
ee
n b
ee
an
d w
asp
re
no
m a
lle
rgy w
ith
sLT
an
d B
AT
th
an
with
ST
or
sIg
E
Cellular Allergen Stimulation Test (CAST) 2003, a review
© 2004 Esmon Publicidad J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273
258
Accordingly, the diagnostic gold standard in foodallergy is usually considered to be the double blind placebo-controlled food challenge (DBPCFC) [31]. However, thisprocedure is quite cumbersome, expensive since usually itrequires some hospitalisation, and may be quite dangerousfor the patient. In addition, DBPCFC should be recognisednot to be entirely reliable [32]. Accordingly, some in vitrotest enabling to replace, at least in part, provocation foodchallenges would be most welcome.
Although theoretically the CAST assay could be ofgreat interest in this indication, only three validated studiesencompassing 151 patients and 79 controls are recorded[30,33] (Table 3). These studies show, however, thatperformance of in vitro food allergen-induced sLT releaseassays may be quite helpful in the diagnosis of foodallergy. Particularly suggestive have been the recent resultsof Ballmer et al. [30] which show that CAST enables todistinguish between patients with immunological cross-reactivity between birch pollen, on the one hand, andcelery, hazelnut and carrot, on the other hand, thosepresenting a clinical reactivity with positive DBPCFCfrom those who do not have been particularly suggestive.
It is obvious that the results are also very muchdependent upon the quality and standardization of theallergen used [32], which is particularly difficult toachieve with food allergens. Accordingly, the use ofrecombinant food allergens [34] is to be encouraged andfurther investigated. It is also likely that combination ofCAST with flow cytometric basophil activation assayswill improve the diagnostic efficiency. Althoughsometimes useful on an individual basis [32,35],histamine release assays have often proven less reliablein food allergy; in particular, patients with food allergiesoften present with high spontaneous histamine release,making the interpretation of the test impossible [36].
Allergy to insect venoms
Several more or less anecdotal studies but at least 7well controlled and validated studies in a total of 351patients with bee and/or wasp venom allergies and 60controls have been published (Table 4). Since diagnosisof IgE-mediated insect venom allergy by skin tests ordetermination of allergen-specific IgE is not entirelyreliable [37-39], it appears that CAST should definitelytake its place in routine diagnosis, at least in thosepatients where skin tests and/or specific IgEdeterminations do not match history. Up to 33% of insectvenom allergy patients proven by provocation (insectsting) have negative skin tests [38,39]. An accuratediagnosis is particularly important to confirm theindication for immunotherapy [40]. In this area also,the combined CAST and basophil activation test appearsto have a particularly high diagnostic efficiency [40,41].
In one field of veterinary allergy, the horse’s summerdermatitis due to allergy to mosquito stings, the CAST
has shown to be the most and only efficient diagnosticmethod [42].
Occupational allergies
Although individual cases with various occupationalallergens have been occasionally reported, most of thepublished experience refers to latex (Table 5).
Among two controlled and validated studies, oneshowed for CAST a sensitivity lower than specific IgEdetermination and skin tests [43], the other one indicatedan acceptable sensitivity (81%) and a particularly highspecificity (97-100%) [44]. Considering that skin testsand specific IgE determinations are also not entirelyreliable in latex allergy [45], cellular tests provide auseful diagnostic complementation, particularly in thisoccupational allergy which may have seriousprofessional consequences. There too, combination ofCAST with flowcytometry has proven particularlyefficient (sensitivity: 93%; specificity: 100 %) [44].
Allergy to drugs
The availability of the CAST assay has since 1993drawn quite a bit of interest, as with the exception ofBetalactam allergy, skin tests and specific IgEdeterminations are seldom available or relevant.Furthermore, it soon appeared that CAST may also yieldinteresting results in some non-IgE mediated pseudo-allergies to drugs, such as NSAIDs [46-49]. However,in the first years, only anecdotal, not well controlledstudies appeared (Table 6a) and it is only in the last fewyears that a large number of well controlled and validatedstudies has been published, particularly on Betalactams,NSAIDs and analgesics. We record at this time 21validated studies encompassing 669 patients and 467controls (Table 6b).
In immediate-type allergy to Betalactams, fivestudies [9,50-53] generally agree that sensitivity is ratherlow, varying between 30 and 50 %, depending also uponthe type of Betalactam allergen used (drug itself orplurivalent drug-polylysine or drug-protein conjugate).Specificity, on the other hand, usually reached 80% ormore. While that kind of sensitivity may well beconsidered too low for an ideal test, it is almost a matterof diagnostic philosophy to declare it useless on a routineor individual basis, as some have done [9]. Particularlyconsidering that the other available tests (skin tests,specific IgE) are also far from being perfect [54], it maybe considered, provided specificity remains high, thatany positive CAST test in drug allergy contributes toconfirm a diagnostic suspicion, while a negative CASTcannot exclude it. In particular, the CAST test has beenfound positive in numerous cases of Betalactam allergyproven by provocation challenge but demonstrating
A.L
. de Weck, et al.
J Invest Allergol C
lin Imm
unol 2004; Vol. 14(4): 253-273
© 2004 E
smon Publicidad
p g
Ref No Author Allergens Patients Nb Result Controls Nb Results Positivity criteria Comments
A. Anecdotal studies
139 Cirla et al, 1995 wheat flour allergic bakers 6 6/6 sLT pos
102 SainteLaudy,1995 latex latex allergics 4 sIgE pos 3/4 sLT pos healthy 2 sLT neg 100 pg/ml > Bckg HR least sensitive
3 sIgE neg 2/3 sLT pos BAT pos also in HR a-IgE neg
B. Validated studies
43 Marais et al, 1997 latex latex allergics 23 sLT SE 43%; sIgE SE56% Healthy 10 patients with urticaria: sLT neg
ST SE 86% -HR SE 45%
44 Sanz et al,2003 latex latex allergics 43 sLT SE 81%, sIgE SE 88% Healthy 30 sLT 97-100% sLT >300 pg/ml; SI >4
ST pos from ROC curves
Table 5. Occupational allergens; Latex.
Table 6A. Drugs.
Ref No Author Allergens Patients Nb Result Controls Nb Results Positivity criteria Comments
A. Anecdotal Studies
7,46 De Weck , 1993 Betalactams allergic certain 9 4/9 sLT pos
dubious 5 1/5 sLT pos
140 Bircher , 1995 Betalactams allergic 8 3/8 sLT pos sLT pos in anaphylaxis only
113 Hippler , 1995 Betalactams allergic 8 8/10 sLT pos Healthy 2 2/2 sLZ neg
Varia allergic 2
47 Brunner,1996 ASA anaphylaxis 9 sLT > in Prov pos healthy 6 no HR to ASA
Analgesics anaphylaxis 6
141 Miyahara,1996 Cefotiam contact urticaria 3 3/3 sLT pos; 3/3 sIgET Healthy 5 5/5 sLT neg
71 Bircher ,1996 Betalactams Anaphylaxis 5 5/5 sLT pos;3/5 sIgE; 5/5 ST Healthy 4 4/4 neg sLT, sIgE, ST
Urticaria 6 0/6 sLT pos; 2/6 sIgE; 5/6 ST
Exanthema 5 1/5 sLT pos;0/5 sIgE; 5/5 ST
Dubious 6 0/6 sLT pos; 0/6 sIgE, 0/6 ST
142 SainteLaudy ,1996 ASA,benzoate allergics 27 12/27 pos ( ASA 8/12;,
dyes benzaote 6/12, dyes 4 & 5 /12)
48 Hippler,1997 Betalactams allergics 14 sLT pos 10/14 Healthy 2 sLT neg 2/2
NSAIDs 6 sLT pos 4/6
varia 10 sLT pos 6/10
143 Sabbah ,1997 Paracetamol allergics 3 3 sLT pos
144 Sabbah,1998 myorelaxants anaphylaxis 16 sLT > in cases of demonstrated 1 sLT pos to Gelofusin
RCM anapyhlaxis
49 Höxtermann ,1997 ASA ASA intolerant 18 9 sLT pos.
127 Leynadier, 1999 lidocaine allergic? 1 sLT false pos
128 Venière et al,2000 various drugs allergic? 22 only 2 CAT and BAT pos 100 pg > Bckg
129 Scherer,2003 Isosulfan blue anaphylaxis 1 BAT pos, sLT neg
259
Cellular Allergen Stimulation Test (CAST) 2003, a review
© 2004 Esmon Publicidad J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273
Tabl
e 6B
. Dru
gs.
Ref
No
Au
tho
rA
llerg
en
sP
ati
en
tsN
bR
esu
ltC
on
tro
lsN
bR
esu
lts
Po
sit
ivit
y c
rite
ria
Co
mm
en
ts
B.
Va
lid
ate
d s
tud
ies
56
,57
Cze
ch
,1
99
5A
SA
, C
5a
AS
A p
rov p
os
8sL
T C
5a
11
88
pg
/ml (6
50
-21
00
)H
ea
lth
y8
sL
T C
5a
37
6 p
g/m
l (1
20
-40
0)
no
sL
T b
y A
SA
(1
0,1
00
ug
/ml)
AS
A p
rov n
eg
7sL
T C
5a
30
9 p
g/m
l (1
15
-45
0)
14
We
di .,
19
96
AS
AA
SA
pro
v p
os
6sL
T C
5a
> c
on
tro
ls (
~2
00
0 p
g/m
l)H
ea
lth
y4
6sL
T C
5a
~2
00
pg
/ml
no
sL
T b
y A
SA
- s
LT
pro
du
ce
d
by b
aso
s,
no
t b
y e
osin
os
12
4M
ew
es ,
19
96
AS
AR
hin
itis
,AS
A
pro
v p
os
8sL
T A
SA
68
6 p
g;
C5
a 2
19
6 p
g/m
lH
ea
lth
y8
sL
T A
SA
34
6 p
g;C
5a
33
4 p
gn
o s
LT
> b
y C
5a
in
co
ntr
ols
Rh
initis
AS
A p
rov n
eg
9sL
T A
SA
20
9 p
g;
C5
a 7
51
pg
/ml
Bckg
sL
T 2
59
pg
AS
A 1
0 u
g/m
l
14
5,1
46
Wo
lan
czyk ,
19
97
AS
AA
sth
ma
AS
A p
rov p
os
15
10
/15
sL
T A
SA
po
s (
SE
66
%)
He
alth
y1
00
/10
sL
T A
SA
po
s (
SP
10
0%
)
Asth
ma
AS
A p
rov n
eg
13
0/1
3 s
LT
AS
A p
os
sL
T A
SA
< 5
0 p
g/m
lsL
T 2
00
pg
/ml >
Bckg
AS
A 2
00
,20
,2 u
g/m
l
65
Cze
ch
., 1
99
4A
dd
itiv
aU
rtic
ari
a a
cu
te8
sL
T C
5a
, fM
LP
> p
atie
nts
in
in
terv
al
He
alth
y8
Urt
icaria in inte
rval
7
14
7K
ub
ota
, 1
99
7A
SA
Urt
ica
ria
,sh
ock
8sL
T A
SA
SE
37
%;
me
an
22
1 p
g/m
lH
ea
lth
y5
sL
T A
SA
SP
80
%;
me
an
13
2 p
g/m
lsL
T 1
00
pg
/ml >
Bckg
Dic
lofe
na
cR
hin
itis
12
sL
T D
iclo
SE
50
%O
the
r d
rug
s5
sL
T D
iclo
SP
90
%S
I >
2
An
tip
yri
n5
sL
T A
ntip
yr
SE
80
%sL
T A
ntip
yr
SP
10
0%
59
,6M
ay
19
99
,20
00
AS
A,
NS
AID
sA
sth
ma
pro
v p
os
22
sL
T S
E A
SA
50
%;+
N
SA
IDs 7
1%
He
alth
y3
0sL
T >
me
an
ato
pic
s +
3S
DA
SA
+C
5a
> A
SA
alo
ne
bu
t
Urt
ica
ria
pro
v p
os
16
sL
T S
E A
SA
81
%;+
N
SA
IDs 1
00
%A
top
ic2
0S
LT
SP
>9
9%
on
ly in
AS
A in
tole
ran
t
24
hrs
blo
od
sto
rag
e O
K
50
Bir
ch
er,
20
00
Be
tala
cta
ms
An
ap
hyla
xis
7sL
T 6
po
ssL
T 2
00
pg
/ml >
Bckg
sL
T p
os in
an
ap
hyla
xis
on
ly
Urt
ica
ria
10
sL
T 0
po
s
Du
bio
us a
llerg
y1
0sL
T 0
po
s
66
Bu
sse
,20
00
AS
A,
be
nzo
ate
Urt
ica
ria
,sym
pto
ma
tic
14
57
% p
rov p
os;
8 s
LT
C5
a p
os
He
alth
y1
5P
rov n
eg
; sL
T C
5a
ne
gsL
T C
6a
> b
y p
rov p
os
me
tab
isu
lfite
sU
rtic
ari
a,a
sym
pto
ma
tic
15
20
% p
rov p
os;
6 s
LT
C5
a p
os
55
Pie
rch
als
ka
,20
00
AS
AA
sth
ma
,AS
A p
rov p
os
26
sL
T B
ckg
84
pg
; A
SA
12
7 p
g/m
lA
SA
18
ug
/ml
Asth
ma
,AS
A p
rov n
eg
32
sL
T B
ckg
82
pg
; A
SA
12
2 p
g/m
lA
SA
> s
LT
in
HI-
60
ce
ll lin
e
58
Ce
lik ,
20
01
AS
AA
sth
ma
AS
A p
rov p
os
12
sL
T B
ckg
35
3 p
g;
AS
A 4
53
pg
He
alth
y1
3sL
T B
ckg
35
8 p
g;
AS
A 4
05
pg
PG
E2
in
hib
its s
LT
re
lea
se
Asth
ma
AS
A p
rov n
eg
13
sL
T B
ckg
35
4 p
g;
AS
A 3
53
pg
9L
eb
el ,2
00
1B
eta
lacta
ms
sL
T S
E 4
3%
30
sL
T S
E 4
3 %
He
alth
y6
4sL
T S
P 7
9%
ratio
ne
t sL
T /
cu
toff
giv
en
sL
T b
ett
er
as H
R b
ut
ind
ivid
ua
l re
su
lts
AS
A1
9sL
T S
E 2
1 %
sL
T S
P 8
8%
by m
ea
n o
f co
ntr
ols
co
nsid
ere
d n
ot
use
ful
Ace
tam
ino
ph
en
6sL
T S
E 3
3 %
sL
T S
P 1
00
%(1
61
, 1
52
,28
5)
14
8W
ed
i ,2
00
0A
SA
, C
5a
Urt
ica
ria
, a
sth
ma
18
sL
T C
5a
SE
83
%;
69
2 p
g/m
lH
ea
lth
y
66
sL
T C
5a
SP
94
%;
11
7 p
g/m
lsL
T C
5a
> 3
00
pg
als
o C
5a
> t
o f
ML
P ,
PA
F
AS
A p
rov p
os
pro
v n
eg
67
Wo
rm ,
20
01
Ad
ditiv
aA
D p
os p
rov
9sL
T A
g S
E 7
8%
He
alth
y
10
sL
T A
g n
eg
( <
10
0 p
g/m
l n
et)
sL
T >
me
an
co
ntr
ols
+ 3
SD
a-I
gE
no
n r
esp
on
de
rs 0
/28
(Tart
razin
e 3
AD
neg p
rov
9sL
T a
-Ig
E :
AD
28
26
pg
; co
ntr
ols
16
89
pg
Be
nzo
ate
4
Nitrite
s 7
,varia 4
)
13
1A
bra
ha
mse
n,2
00
1C
5a
, a
-Ig
EA
sth
ma
AS
A p
rov p
os
10
sL
T C
5a
14
.4 p
g /
10
x5
ce
llsH
ea
lth
y9
sL
T C
5a
7.5
pg
/ 1
0x5
ce
llsC
5a
do
es n
ot
dis
tin
gu
ish
AS
AC
5a
/ a
-Ig
E s
LT
ra
tio
dia
gn
ostic f
or
AS
AA
sth
ma
AS
A p
rov n
eg
12
sL
T C
5a
22
.9 p
g /
10
x5
ce
llsp
rov p
os o
r n
eg
asth
ma
tics
intr
insic
asth
ma
- sL
T p
rod
uce
d b
y b
aso
s
Alle
rgic
asth
ma
(Ig
E)
7sL
T C
5a
9.6
pg
/ 1
0x5
ce
lls
51
,52
Sa
nz ,
20
00
,20
02
Be
tala
cta
ms
BP
/AX
ST
po
s2
3sL
T S
E 3
5%
; +
BA
T :
SE
76
%H
ea
lth
y
30
sL
T S
P 8
3%
sL
T 1
00
pg
/ml >
Bckg
op
tim
al S
E b
y c
om
bin
atio
n s
LT
+ B
AT
AX
ST
po
s3
4sL
T S
E 3
2%
; +
BA
T :
SE
61
%S
T n
eg
BA
T S
P 9
3%
BP
/AX
ST
ne
g1
6sL
T S
E 4
7%
; +
BA
T:
47
%
53
Me
dra
la,
20
02
Be
tala
cta
ms
Imm
ed
iate
typ
e3
3sL
T B
P S
E3
0%
He
alth
y1
3
alle
rgy
sL
T P
PL
SE
18
%
C
om
bi A
g 7
4 %
ST
ne
g
sL
T B
PO
-HS
A S
E 5
0%
64
Ga
mb
oa
,20
03
Dip
yro
ne
An
ap
hyla
xis
15
sL
T S
E 5
2%
; B
AT
SE
42
%O
the
r a
llerg
ies
30
3 s
LT
po
s ;
SP
90
%sL
T 1
00
pg
/ml >
Bckg
; S
I >
3o
ptim
al S
E b
y c
om
bin
atio
n s
LT
+ B
AT
+
ST
Urt
ica
ria
11
sL
T +
BA
T S
E 7
6 %
Pro
v n
eg
BA
T S
P 1
00
%
14
9S
ch
äfe
r,1
99
9A
SA
asth
ma
tics,
10
AIA
,1
8sL
T >
, P
GE
2 <
in
AIA
he
alth
y1
0n
o >
sL
T>
sL
T in
AIA
in
vitro
an
d in
viv
o (
Pro
vo
c)
8 A
TA
61
Sa
nz e
t a
l, 2
00
3A
SA
+ 4
NS
AID
sA
sth
ma
P
rov p
os
60
AS
A s
LT
SE
2
2%
He
allt
hy
30
AS
A s
LT
SP
89
%sL
T 1
00
pg
/ml >
Bckg
SI>
2P
rov p
os w
ith
in 1
mo
nth
: S
E 8
8%
Urt
ica
ria
4 N
SA
IDs S
E 4
8%
Pro
v n
eg
4 N
SA
IDs S
P 6
8%
11
0F
ari
a,2
00
3A
SA
asth
ma
to
AS
A1
5o
nly
1/1
5 s
LT
po
sa
top
ics
92
/9 s
LT
po
ssL
T 1
80
pg
> b
ckg
CA
ST
no
t u
se
ful
urt
ica
ria
to
AS
A1
41
4 s
LT
ne
gh
ea
lth
y3
3 s
öT
ne
g
15
0P
ere
ira
-Sa
nto
s,
20
02A
SA
, D
icu
rtic
ari
a t
o N
SA
IDs
37
4 s
LT
po
s/6
Pro
v p
os
resp
alle
rgy
20
all
sL
T n
eg
co
rr C
AS
T p
os/A
SA
pro
v p
os p
=<
0.0
1
Pro
v n
eg
= s
LT
ne
gh
ea
lth
y1
5a
ll sL
T n
eg
260
A.L. de Weck, et al.
J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273 © 2004 Esmon Publicidad
261
negative skin and specific IgE tests [52]. Taken on anindividual patient’s basis, CAST may therefore oftenprovide some useful information.
In the case of pseudoallergic reactions to aspirin andother NSAIDs, no other diagnostic in vitro test has beenavailable before CAST. Among the ten controlled andvalidated studies published so far (Table 6b), one iscompletely negative [55], one because of a sensitivity ofonly 21% despite a specificity of 88% leads to uselessness[9] and the other eight studies report on diagnosticefficacy, with sensitivities varying between 60 and 71%and specificities between 97 and 100%. However, threeof these studies report positive results only with C5a andnot with ASA [56-58] and two report that use of otherNSAIDs beyond ASA alone considerably improvessensitivity [59,60]. Heterogeneity in the allergens used,particularly in the dose, may possibly explain theheterogeneity in results; for example, the only totallynegative study [55] used a single dose of ASA almost 20-50 times lower than most other studies. In the case ofpseudoallergy to NSAIDs also, combination of CASTwith flowcytometry may improve diagnostic efficiency[61]. In any case, the classical statement often repeatedover 20 years [62] that no diagnostic in vitro tests exist inpseudoallergy to NSAIDs is certainly no longer valid.Our point of view is reinforced by the recent report thatincreased release of 15-HETE by leukocytes uponincubation by ASA is also detected in asthmaticsintolerant to aspirin [63].
Another area in which CAST has recently shown tobe useful is in IgE-mediated allergy to analgesics, e.g.Dipyrone ( Metamizol ) [64].
Finally, a few studies point to the usefulness of CASTin allergic or pseudoallergic reactions to food additivessuch as dyes or preservatives [65-67]. Such reactions arerelatively rare and difficult to confirm clinically byprovocation tests, because of the usual multiplicity ofsuspected substances. However, the frequent success ofavoidance diets in chronic urticaria (68) indicates thatadditives may exhibit causal or at least adjuvant effectsmore often than usually believed. Since CAST showssometimes positive results and has a high specificity, andsince no other tests are reliable, it seems that the CASTtest would be worth carrying out before provocation tests.
In summary, despite a suboptimal sensitivity, theCAST test has definitely shown to be helpful in diagnosisof immediate-type allergies and pseudo-allergies todrugs, particularly when skin tests or determination ofdrug-specific IgE are not available. In delayed-typeclinical reactions, on the other hand, e.g. morbilliformexanthema, which are caused by drug-specific Tlymphocytes [69,70], the CAST assay is mostly negative[71].
Immunotherapy follow up
Immunotherapy with aeroallergens for allergic
asthma and/or rhinitis has been shown by many studiesto have beneficial effects on clinical symptoms of allergy[72]. Its mechanisms of action are still the object ofdiscussion and hypotheses. Some of the majorimmunological effects are a shift in the balance ofallergen-specific Th1 and Th2 lymphocytes and astimulation of T regulator lymphocytes, withcorresponding changes in the pattern of allergen-inducedcytokine production [73]. The diminution of allergicsymptoms upon allergen exposure, however, is probablymore directly related to an impaired release ofinflammatory mediators, such as histamine andsulfidoleukotrienes, that are responsible for thesesymptoms. Indeed, several authors have reported adecrease of allergen-induced histamine release fromblood basophils following immunotherapy [74].
It was therefore logic to investigate with the CASTassay whether the beneficial effects of classicalimmunotherapy, which are often demonstrated by moreor less subjective criteria, can be effectively andobjectively confirmed by an in vitro test.
Several studies (Table 7) have attempted to answerthat question, particularly in the field of inhalationallergy to house dust mites and to pollens [75-78] andinsect venoms [79-84]. In insect venom allergy,although a diminution of sLT release is sometimesobserved following immunotherapy, the correlationbetween in vitro results and clinical benefit is notimpressive [83] and monitoring of venomimmunotherapy usually considered not useful. Ininhalation allergies, when clinical symptoms havedecreased by more than 50% followingimmunotherapy, a corresponding decrease in SLTrelease in vitro is consistently observed [75,78]. Themost impressive results reported to date are thosefollowing immunotherapy combined with anti-IgEtherapy [85]. In that case, the diminution of allergen-induced sLT release in vitro was highly significant andcorrelated with clinical benefit.
More follow up studies, possibly with moresophisticated techniques (e.g. with inclusion of allergendose-response curves, repeated tests at 3 monthsintervals, etc.) to document the pathophysiologicalsuccess of immunotherapy regimens would certainly bedesirable.
Drugs and biological factors affectingsLT production
In a few published studies CAST has shown (Table8 A) to be suitable for pharmacological studies of drugsaffecting basophil reactivity and mediator release, suchas antihistamines [21], steroids [86,87], ambroxol [88]or various chemicals [89]. In pharmacological researchon potential anti-inflammatory drugs, particularly indrugs affecting leukotriene production, the CAST assay
Cellular Allergen Stimulation Test (CAST) 2003, a review
© 2004 Esmon Publicidad J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273
may be quite useful, but up to now suchresults from the pharmacological industryhave rarely been published.
In the literature, a number of isolatedstudies document the effect of biologicalfactors such as cytokines (90-93) onleukotriene release. Antibodies, such assecretory IgA [94] and particularly anti-IgE and/or anti-FcER1 antibodies, asencountered in many cases of chronicidiopathic urticaria, have been shown toinduce sLT production [95,96]. Sincesome of these antibodies do not inducesymptoms and mediator release, the CASTassay with autologous serum couldbecome an important diagnostic tool inchronic urticaria.
Correlation of CAST withclinical reactivity and otherdiagnostic tests
From a practical point of view, it isimportant to evaluate the diagnostic valueof CAST in respect to the clinical allergicsymptoms and to the other diagnostic testsavailable such as skin tests, specific IgEdetermination ( e.g. CAP assay) and othercellular tests.
Relationship to nature andseverity of the clinical allergicreaction
Since CAST detects the capacity ofbasophils to release sLTs as mediators ofallergic inflammation, thereby expressingcellular reactivity [7,97], which is notobligatorily proportional to sensitisationmanifested by the presence of IgEantibodies, one could theoretically expectbetter correlation of CAST with theseverity of clinical symptoms. However,there are not many data available on thistopic.
In allergic rhinitis, a good correlationbetween CAST results and the severity ofreaction to allergen challenge provocationhas been reported [21]. In asthma, on theother hand, correlation between asthmaseverity and the level of CAST positivityin sLT pg/ml is rather poor [7,98]. Thesame has been reported for in hymenopteravenom allergy [99,100]. In allergic clinicalreactions involving primarily local tissueTa
ble
7. F
ollo
wup
Im
mun
othe
rapy
.p
py
Ref
No
Au
tho
rA
llerg
en
sP
ati
en
tsN
bR
esu
ltC
on
tro
lsN
bR
esu
lts
Co
mm
en
ts
11
7C
osa
ch
ov,1
99
5G
P,r
ag
we
ed
Asth
ma
ato
pic
s6
sL
T d
imin
ish
ed
fro
m 5
61
pg
/ml to
19
6 p
g/m
l a
fte
r
Mo
uld
s6
mo
nth
s I
T (
gro
up
me
an
)
75
De
We
ck ,
19
95
HD
MIT
HD
M1
6sL
T d
im >
50
% in
50
% o
f p
atie
nts
IT
12
mo
nth
s
IT P
lace
bo
14
no
ch
an
ge
sL
T in
pla
ce
bo
tre
ate
d
13
9C
irla
, ,1
99
5w
he
at
flo
ur
IT6
sL
T A
g 5
58
/ml p
g t
o 1
71
pg
/ml a
fte
r 3
mo
nth
s I
T
sL
T a
-Ig
E 2
14
1 p
g/m
l to
14
16
pg
/ml
76
Fe
rre
r ,
19
98
HD
Ma
llerg
ics I
T 2
.5 y
rs3
5sL
T H
DM
13
20
pg
/ml
alle
rgic
s n
o I
T5
7sL
T H
DM
20
28
pg
/ml
sL
T a
-Ig
E w
ith
IT
20
86
pg
/ml
GP
alle
rgic
s I
T 2
.5 y
rssL
T G
P 1
88
4 p
g/m
la
llerg
ics n
o I
T1
5sL
T G
P 2
21
3 p
g/m
lsL
T a
-Ig
E n
o
IT 1
33
9 p
g/m
l
HD
M +
GP
79
Bu
sse
, 1
99
6ve
no
ms
80
Ju
tel ,
19
96
ve
no
ms
Ultra
-ru
sh
IT
(3
-6 h
rs)
7n
o <
sL
T b
ut
< t
ota
l H
Rno o
bvio
us d
ecre
ase s
LT
81
Eberlein
, 1997
venom
s
77
Me
dra
la,1
99
7G
PIT
clin
su
cce
ss
8sL
T G
P b
efo
re I
T 5
01
pg
,aft
er
IT 3
86
pg
, a
fte
r se
aso
n 6
15
pg
/ml
de
cre
ase
sL
T o
nly
in
pa
tie
nts
IT c
lin f
ailu
re9
sL
T G
P b
efo
re I
T 8
24
pg
,aft
er
IT 1
05
7 p
g,
aft
er
se
aso
n 7
97
pg
/ml
clin
ica
lly r
ea
ctin
g t
o I
T
78
Sa
raçla
r,1
99
7H
DM
alle
rgic
s u
nd
er
IT1
3sL
T <
50
% in
5/6
pa
tie
nts
with
sym
pto
ms <
50
% a
fte
r O
Za
llerg
ics,
no
IT
5sL
T u
nch
an
ge
d<
na
sa
l p
rov s
ne
eze
s 5
3%
aft
er
IT
(12 m
o)
82
Pie
rkes,1
999
wasp v
enom
syste
mic
alle
rgy
22
< s
LT
and H
R a
fter
rush IT
(1 w
eek)
< s
LT
du
e t
o I
L-1
0 ,
IF
N-g
am
ma
ST
po
s,
sIg
E p
os
83
,99
Bo
relli
, 1
99
9,2
00
3b
ee
,wa
sp
be
e a
llerg
ics
20
aft
er
3 y
rs I
T:
23
ST
dim
inis
he
d,
on
ly 2
CA
ST
be
co
me
ne
gC
AS
T n
ot
use
ful to
pre
dic
t
ve
no
ms
wa
sp
alle
rgic
s2
31
6 p
rovo
c.
ne
g a
fte
r IT
; a
ll re
ma
in C
AS
T p
os
ou
tco
me
of
IT
84
Szym
an
ski, 1
99
9b
ee
19
alle
rgic
ST
,sIg
E p
os
43
sL
T m
ae
n ~
50
00
be
fore
IT
sL
T >
60
0 in
10
/11
pa
tie
nts
wa
sp
18
pse
ud
o S
T,s
IgE
ne
g1
1sL
T m
ea
n ~
15
00
aft
er
ITp
se
ud
oa
llerg
ic (
ST
,sIg
E n
eg
)
VIT
failu
re7
85
Ko
pp
,20
02
GP
rh
initis
(6
-17
yrs
)2
3IT
GP
+ a
nti-I
gE
Ab
: s
LT
4
16
pg
/ml
GP
IT
alo
ne
2
4sL
T
24
90
pg
/ml
IT
< s
LT
als
o t
o A
g n
ot
use
d f
or
IT
Bir
ch
2
3IT
bir
ch
+ a
nti-I
gE
Ab
: s
LT
20
7 p
g/m
lB
irch
IT
alo
ne
22
sL
T
24
89
pg
/ml
262
A.L. de Weck, et al.
J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273 © 2004 Esmon Publicidad
263
Tabl
e 8.
Eff
ect o
f va
riou
s fa
ctor
s on
sLT
pro
duct
ion.
p
Ref
Au
tho
rA
llerg
en
Dru
gP
ati
en
tsE
ffect
Co
mm
en
ts
A. D
rug
s
21
Ka
layci ,1
99
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itis
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by A
g,
no
t b
y a
nti-I
gE
n
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ha
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er
pla
ce
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ttt
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r)
10 m
g/d
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ays
86
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cke
r ,1
99
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alle
rgic
sn
o e
ffe
ct
of
Be
ta a
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t o
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se
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ti-s
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on
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ele
ase
87
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cke
r ,1
99
7G
P,H
DM
ste
roid
sA
llerg
ics
10
No
eff
ect
of
ste
roid
s in
vitro
on
sp
on
tan
eo
us
> s
po
nta
ne
ou
s s
LT
re
lea
se
in
se
aso
n
sL
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ele
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t <
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-pri
me
d c
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y ionom
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19
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n a
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r ce
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/80
ma
st
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lls b
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for
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89
Str
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cre
ase
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s
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se
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olli
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sis
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er
15
min
pre
inc w
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N
Alle
rgic
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92
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ka
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98
LP
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leb
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llerg
ic a
sth
ma
19
incre
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62
pg
/ml
10
he
alth
y c
on
tro
ls :
sL
T 1
91
pg
/ml
CO
PD
9n
ot
sig
nific
an
t in
cre
ase
sL
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47
4 p
g/m
la
sth
ma
tics >
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T t
o L
PS
15
1T
askila
, 2
00
0n
ett
leco
nta
ct
urt
ica
ria
94
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ra ,
19
98
se
cre
tory
Ig
Ah
ea
lth
y d
on
ors
9sL
T ,
HR
by im
mo
bili
ze
d s
IgA
prim
ing
eff
ect
of
IL
-3 a
t 5
-50
pg
/ml
93
We
hn
er,
20
01
Sta
ph
.au
reu
sa
top
ic d
erm
atitis
15
Bckg
: 7
pg
; IL
-3 1
56
pg
: S
tap
h 1
07
pg
/ml
9 h
ea
lth
y :
Bckg
7 p
g;
IL3
7 p
g:
Sta
ph
17
pg
en
tero
toxin
ssIg
E S
tap
h p
os
Sta
ph
+ I
L-3
: 2
80
2 p
g/m
lS
tap
h +
IL
-3:
17
pg
- s
IgE
Sta
ph
ne
g
95
We
di .,
20
02
au
tolo
go
us
ch
ron
ic u
rtic
ari
a,S
T p
os
20
incre
ase
d s
LT
to
au
tolo
go
us s
eru
m in
10
he
alth
y,
10
ato
pic
co
ntr
ols
, n
o s
LT
se
rum
ch
ron
ic u
rtic
ari
a,S
T n
eg
20
ind
ivid
ua
l ca
se
s o
f b
oth
gro
up
ssL
T in
hib
ite
d b
y d
ap
so
ne
, ch
loro
qu
ine
sL
T c
orr
ela
ted
with
urt
ica
ria
se
ve
rity
lido
ca
ine
, m
izo
lastin
e
96
Fe
rre
r ,
20
02
au
tolo
go
us
ch
ron
ic u
rtic
aria
60
so
me
se
ra c
au
se
sL
T ,
HR
50
co
ntr
ols
, n
o s
LT
seru
m
Cellular Allergen Stimulation Test (CAST) 2003, a review
© 2004 Esmon Publicidad J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273
mast cells, it is understandable that a test reflecting thereactivity of blood basophils may not be closelycorrelated. This might be different in generalized allergicreactions, such as anaphylactic shock. Indeed, it has beenreported in Betalactam allergy that CAST is positiveonly in cases of anaphylaxis [50,71] but this has notbeen confirmed by other authors, who find CASTpositive also in cases of generalized urticaria [51,52]. Acorrelation of CAST with anaphylaxis has also beenreported for foods [33]. There is evidence that sLTs arereleased in vivo during anaphylactic reactions [101].
Correlation with other cellular tests
The histamine release test has been used in allergyresearch since over 40 years but has seldom been appliedroutinely to allergy diagnosis (2) mainly because it isrelatively cumbersome and not very sensitive. In principle,however, it is quite similar to the CAST assay. Both testshave been compared by several authors in clinicalsituations, particularly for inhalant allergies [98,102,103],insect venom allergies [37,104,105], food allergies[33,34,36,106] as well as hypersensitivity to NSAIDs[57,102] and other drugs [63,66]. For anti-IgE stimulantand protein allergens, correlation has usually been foundto be rather good but this has not been the case for haptensand drugs (Fig 1). In general, histamine release has beenfound to be less sensitive than CAST [50]. An additionaldrawback is that histamine release may be non specificallyhigh and false positive, in case of allergen cytotoxicity and/or of recent in vivo allergen exposure [36].
There are a number of situations (e.g. drugs, nonspecific reactions to some basophil stimulants) in whichhistamine and sLT release may be dissociated, since bothrest on different intracellular mechanisms [61,107].Dissociation between histamine and sLT release has alsobeen observed on bee venom allergy [37,104,108].
Correlation with skin tests
For most protein allergens such as inhalants [7,18,98]and insect venoms [37,104,105, 107], there is aquantitative correlation between skin tests and CASTresults but this correlation is usually not very high, inthe range of r= 0.4 – 0.6. It has also been reported that anumber of patients allergic to drugs [51,52], insectvenoms [108,109] or latex [43] may show positive CASTdespite a negative skin test. In a number of instances, e.g.hypersensitivity to NSAIDs and non-IgE mediatedallergies, for which no skin tests exist, CAST offers avaluable in vitro approach to diagnosis. In such cases, apositive CAST result may be meaningful but a negativeresult never permits to exclude allergy or hypersensitivity.
Correlation with allergen-specific IgE
In all instances where basophil reactivity and
mediator release to allergens in vitro is based on thepresence of cell-bound specific IgE, a correlation withthe detection of allergen-specific serum antibodies (e.g.CAP assay) should be expected. Indeed, in someinstances, particularly in inhalation allergies to proteinallergens, such correlation has been found. Thequantitative correlation, however, is not very high inmost instances (r = 0.20 – 0.72). While the presence ofallergen-specific serum IgE merely reflects sensitisation,basophil mediator release reflects additional parameterssuch as IgE avidity and cellular reactivity [7,15].
In several instances, particularly in allergy toBetalactam antibiotics, determination of allergen-specific serum IgE has been found to be less sensitivethan CAST [51,52].
Complementarity of CAST with flowcytometric basophil activation
As already indicated, the combination of sLTdetermination by ELISA and of basophil activation byflowcytometry, as commercially available in the CASTCombi test, may be of great diagnostic benefit, as alreadyvalidated for Betalactam [51,52], NSAIDS [61],metamizol [64] and latex [110] allergy. Following a singleincubation of buffy coat leukocytes stimulated byallergens in vitro, this test combines the flowcytometricanalysis of CD63 expression on basophils with thedetermination of sLTs by ELISA on the cell supernatant.
One could have thought that both tests would run inparallel and show high correlation. As a matter of factthis is true for high levels of hypersensitivity and proteinallergens (Fig. 1). But it is not quite true for weak levelsof stimulation, as frequently encountered with drugs.In that case, the combined performance of both testsmarkedly increases sensitivity without significantlydiminishing specificity (Fig.1). The correlation betweenFAST and CAST becomes markedly poorer with drugsstimulating basophils by non specific mechanisms, likeNSAIDs. For allergies and pseudoallergies to drugs, aswell as for food allergies, the combined test is certainlyrecommended.
Technical considerations affecting theinterpretation of results
A mere review of published results might bemisleading if a number of technical factors which mayaffect the results were not discussed. Indeed, as isapparent from the literature and from personalexperience, a number of technical factors may beresponsible for discrepant results and many publications,unfortunately, are not sufficiently explicit in that respect.
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A
r = 0.74 n = 120
Correlation FAST CAST anti-IgE
0
1000
2000
3000
4000
5000
6000
7000
0 20 40 60 80 100
Basophil activation %
SL
T p
g / m
lB
0.79 n = 177
Correlation FAST CAST in latex allergy
0
500
1000
1500
2000
2500
3000
0 20 40 60 80 100
Basophil activation %
sL
T p
g /
ml
C
r = 0.20 n = 84
Correlation FAST CAST for Naproxen
0
100
200
300
400
500
600
700
800
900
1000
0 10 20 30 40 50 60
Basophil activation %
sL
T p
g /
ml
D
r = 0.56 n = 120
Correlation FAST CAST for ASA
0
100
200
300
400
500
600
0 10 20 30 40 50 60
Basophil activation %
sL
T p
g /
ml
Figure 1. Correlations between FAST (basophil activation) and CAST (pg SLT/ml) for positive anti IgE controls(panel A), allergy to Latex (panel B), hypersensitivity reactions to Naproxen (panel C) and hypersensitivity reactionsto acetylsalicylic acid ASA (panel D).
Various technical aspects have been discussed elsewhere[7, 8,15]. The good inter- and intra-assay reproducibilityhas been confirmed by several authors [19,25,36,111,112].
Allergen dose
With most protein allergens (e.g. pollen house dustmites, foods), the allergen doses found to yield positiveresults in allergic patients vary between 1 and 100nanograms protein/ ml [7,15,18,19,98]. Since mostallergens used are not standardized in terms of theircontents in molecularly defined allergens, quantitative
comparisons are indeed difficult, particularly, as maybe the case for immunotherapy follow up, when the testis repeated at different times with different allergenbatches. On the basis of histamine release assays, whichtypically show a bell shaped dose response curve, it hasbeen argued [12] that the use in routine testing of a singleallergen dose in CAST is not acceptable. This may betrue if absolute quantitative comparisons were requiredbut in terms of segregation between positive or negativeCAST response, experience has shown that a single doseof 10-20 ng protein almost always enables todiscriminate, at least for aeroallergens and foods. For
FAST CAST correlation anti-IgE FAST CAST correlation in latex allergy
FAST CAST correlation for Naproxen FAST CAST correlation for ASA
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most protein allergens, the CAST dose-response curvehas a very broad bell shape over 3-4 potencies [15,113].Accordingly, and in contrast to what is stated in a recentposition paper [12], a single concentration of allergen(usually 1 – 100 ng/ml final concentration) will sufficeto distinguish between negative and positive cases. Forinsect venoms, on the other hand, which containssubstances causing non specific basophil stimulation athigher doses in healthy controls, the use of at least twoallergen concentrations is beneficial [37,104], althoughnot essential [99]. In drug allergy also, the routine useof two allergen doses, usually in a ratio of 1:5 or 1:10has been found to increase sensitivity [52,61,64]. Theproblem of optimal allergen concentrations for CASTwill probably become simplified by the future increaseduse of recombinant allergens [34].
Spontaneous sLT production
In the CAST assay, nonstimulated leukocytes usedas negative control, seldom yield more than 200 pg sLT /ml, usually markedly less, although some authors reportas much as 17% of random patients showing a nonstimulated background of > 300 pg sLT / ml (114).Consistently higher negative controls in an experimentalseries should raise the suspicion of some technicalmishap, such as the use of endotoxin-contaminatedwater, of non tissue culture grade plastic tubes forincubation or performance of the test in the open airand not in airflow sterile hoods (accidentalcontamination by aeroallergens!). Such disturbingfactors are usually easy to identify.
The interpretation of data from individual patientsshowing an elevated non-stimulated control is moredifficult. Such instances have been observed withvariable frequency; i.e. l 200 – 300 pg/ml in 5-10% insome series [61,115] but as much as 17.9% [28/145] with>300 g/ml in others [114]. The usual interpretation ofsuch findings is that patient basophils have beenstimulated in vivo shortly before blood sampling byexposure to allergen(s) or to some endogenousinflammatory agent (e.g. infection). The increase ofspontaneous sLT production (and /or histamine release)ex vivo during and shortly after the pollen season[18,19,116] or in food allergy where hidden allergenexposure and cross reactivities are probably frequent[33,36] are well documented. Even in cases with anabnormally high spontaneous sLT production andnegative control, interpretation of the test remainspossible if the sLT released upon stimulation by theputative allergen is markedly higher (stimulation index> 2 or 3 ).
Occurrence of non releasersIt is essential to perform also a positive control, to
ensure that the cells used are viable and that thestimulation conditions permit the production of SLT. As
well known and documented, the releasability ofmediators by basophils is eminently variable [7,117] andinfluenced by many factors [97,118]. As standard, IgE-related basophil activation stimulants, anti-IgEantibodies and later anti-IgE Fc R
I antibodies have been
used in the CAST assay as positive controls [7,13].Nevertheless, some patients behave as “non releasers”and the negative data obtained upon allergen stimulationare then difficult or impossible to interpret. Morequestionable are positive results obtained with someputative allergen or ligand, while the anti-IgE inducedcontrol stimulation remains negative. With the histaminerelease assay, the percentage of non releasers may bequite sizeable in some studies: up to 20 – 25 % [2,36].In CAST, the percentage of non releasers with anti-IgEwas usually much lower, below 10% [55,58] and evenbelow 3-5% when using anti-IgE FcR
I [7,26,99,115],
although some series report as much as 19% of thepatients releasing less than 500 pg sLT /ml [114].
Other non specific basophil stimulants may be usedas positive controls, such as fMLP, ionomycin orconcanavalin A. These stimulants, however, are nonspecific, often also act on other cells than basophils andtest stimulation cellular pathways which are different fromthose triggered by IgE. They are therefore, theoreticallyat least, not strictly controls for IgE-mediated allergenstimulation but merely a cell functionality test.
Positivity criteria
A major problem in comparing and interpreting datafrom the literature is that positivity criteria used byvarious authors, including those recommended by themanufacturers, have often been quite different and havevaried with time. Quite frequently, results of allergenstimulation have been considered positive whenindicating 200 pg sLT/ ml above the negative control(or background) [104,119] but for weak stimulations,such as with drugs, particularly NSAIDs, the optimalcutoff to achieve optimal sensitivity and specificity hasbeen 100 pg/ml above background [52,64]. However,in order to account for cases of relatively highbackground, an additional criterion has beenrecommended, namely a stimulation index (specificstimulation / negative control) higher than 2. Lately,optimal cutoffs have been checked for most allergensby ROC curves [51].
Another way to define positivity has been to set up themean of sLT pg/ml induced by allergen in a group ofclinically negative controls and to accept as positive valuesthose exceeding 2 or 3 SD from that mean [37,99,104,112].Since, particularly with drugs, the mean of the control groupmay be quite variable, this mode of calculation was foundto be rather complicated and sometimes misleading.
In any case, it is quite important that published CASTstudies in the future clearly indicate the positivity criteriaused.
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Role of chronic allergen exposure
Theoretically, it could be expected that chronicallergen exposure may affect the results of cellular tests,in particular CAST, in two ways. On the one hand,basophils stimulated in vivo by allergens may continueto release sLT apparently spontaneously in vitro andcause high negative controls. Indeed, this has beenobserved in grass pollen allergics during the pollenseason [18,116] despite a recent contradictory reportusing the FAST test [120]. Another possible effect ofchronic allergen exposure is to render basophils nonspecifically hyperreactive to various stimulants, asobserved also in atopic dermatitis and in house dust miteallergy. Such an effect appears to be the rule in NSAIDhypersensitivity, where the cause for basichyperreactivity is probably some endogenous pro-inflammatory affection [61]. But it has also beenobserved in reactivity to C5a: patients with latex allergyor drug allergies are in part hyperreactive to C5a, whichis not at all the case for insect venom allergics (T.Jermann, unpublished). The formal proof that thishyperreactivity is linked to chronic allergen exposure,however, is still missing.
Role of IL-3 priming
The fact that priming or simultaneous addition ofIL-3 considerably increases the sLT release induced byallergens has been reported more than 15 years ago[97,118] and has been used since the beginning in theelaboration of the CAST assays as used in routinediagnostic practice [7]. The practical importance ofadding IL-3 to obtain adequate sensitivity has beenconfirmed [26,121]. The potentiating effect of IL-3seems more intense for sLT production than for histaminerelease. However, there has also been some opposition,claiming that IL-3 alone, even at the low concentrationrecommended [122,123] may stimulate basophils andcause false positive, non specific reactions. Thiscontroversy has been recently revived, suggesting, forexample that the addition of 2 ng/ml IL-3 increases by70% the degree of background basophil activation [123].While it may be true that the addition of IL-3 slightlyincreases the background, this comes up on a very lowlevel and is not significant in view of the positivity criteriausually chosen. In any case, particularly in drughypersensitivity, the addition of IL-3 often appearsessential for obtaining adequate sensitivity and does notseem to affect specificity. Basophils from atopic donorsseem more sensitive to IL-3 priming than normals[15,119].
Role of C5a activation
C5a is a well known non specific activator ofbasophils [97] and low non stimulating concentrations
of C5a have been thought to enhance the stimulationinduced by specific allergens [59,60]. This has enticedsome authors to use C5a, either alone or together withallergens, to perform diagnostic CAST assays[48,56,57,110,113].
In fact, it does not appear that C5a at concentrationsof 10-8 M and below, really augments the reactivity ofnormal “healthy” basophils [59,124]. This may bedifferent, however, when the cells already possess, tostart with, some state of non specific hyperreactivity,which may be the case in patients with chronic urticaria[125], severe atopic dermatitis [126], asthma andparticularly hypersensitivity to NSAIDs [56,57,61,110].In such cases, C5a alone at the concentration of 10-8M,may already induce basophil activation and sLT release[49,57,110,127-129]. At the higher concentration of 10-7 M.in various categories of allergic patients, C5a aloneinduces significant sLT release in about 50% of the cases(T. Jermann, unpublished).
The general recommendation at present is not to useC5a as an “adjuvant” but it may be interesting toestablish, from a C5a dose-response curve, whether thepatient possesses hyperreactive circulating basophils atthe time of blood sampling.
Cellular origin of sLT in blood tests
SLTs may be produced by different circulating bloodcell type, basophils, eosinophils, monocytes and platelets[130] and therefore it may be asked which cells arecontributing to the sLT release induced by allergens inwhole blood or in isolated leukocytes. The capacity toproduce sLT, when induced by a non specific stimulantsuch as ionophore (A 12387) or fMLP, is about 100-fold lower for eosinophils than for basophils ormonocytes. Experiments performed on isolatedbasophils and /or on basophil-depleted cells have clearlyshown that the main providers of sLT upon IgE-mediatedblood cell activation are the basophils, to an extent ofmore than 90% [97]. This seems also to be the case inhypersensitivity to NSAIDs, which is not mediated byspecific IgE [131].
Use of passive sensitisation with serum
Since the induction of sLT release by basophils fromallergic patients implies interaction of allergens withspecific IgE on the cell surface, it may be expectedthat the same IgE antibodies, when passivelytransferred to normal basophils, would also cause sLTrelease in the presence of allergens. This has indeedbeen used with CAST, e.g. using sera from food [36]or drug [132] allergic patients and a mixture of normaldonor basophils. This has some potential practicalapplications, since it is not always possible to send indue time the patient’s leukocytes to the laboratoryperforming the CAST assay. However, it seems that
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© 2004 Esmon Publicidad J Invest Allergol Clin Immunol 2004; Vol. 14(4): 253-273
this method is less sensitive than when using patientcells. In a group of food allergic patients, the CASTtests performed after passive sensitisation was onlypositive when the serum used contained at least 3.5kU IgE/L of specific IgE [36]. But the passivesensitisation method can also be used across somespecies, e.g. to detect dog IgE with human basophils[133].
Effects of blood storage
The need to use fresh blood or isolated leukocytesis the main obstacle to the routine use of cellular testsin allergy diagnosis. Investigations on the possibilityto keep the blood for several hours or days after bloodsampling have shown that the optimal conditions forkeeping or sending the blood are the following: 24hours at room temperature, suspension in ACDmedium. Under such circumstances, performance isalmost similar to that of fresh cells [7,15,36].
Avoidance of drug therapy before bloodsampling
Several groups of drugs are known to impair basophilreactivity and mediator release in vitro and in vivo. Someantihistamines have been shown to impair the reactivityof ex vivo basophils [21]. Accordingly, it is usuallyrecommended that patients should avoid to take anti-histamines for at least 3 days (4 weeks in the case ofastemizol) before blood sampling in view of a CASTassay.
For corticosteroids, the recommendations shouldbe more differentiated. Unfortunately, the dataavailable are insufficient. This matter is important inpractice, since many severely allergic patients,particularly asthmatics under steroid therapy, cannotinterrupt treatment merely for a diagnostic test.Glucocorticoids in vitro have been reported to indeedinhibit allergen- or non specific stimulant- inducedbasophil activation and mediator release in a dose-dependent manner [21]. Orally administered steroidsappear to interfere with ex vivo histamine and/or sLTrelease [86] but the time required for full recovery ofbasophil reactivity after treatment interruption is notprecisely known. It is usually recommended to abstainfrom oral steroid therapy for 8 days before bloodsampling, but this may not always be possible. Theeffect of oral steroids on CAST is probably alsodependent upon the dose and duration of steroidtherapy. Despite oral steroid therapy, 15 from 22patients still had positive CAST tests to NSAIDs [59].
For steroids administered by inhalation, which is thecase for a large majority of patients with allergicrhinosinusitis or asthma, the evidence that treatmentcould interfere with the CAST assay is not obvious [55].In some studies, it appeared that the duration of the
steroid effect on ex vivo basophil mediator release wasabout 14 days [59]. A more formal investigation on theeffect of inhaled steroids on sLT release, however, couldnot be found in the current literature.
Conclusions
During the past 10 years, the CAST assay has beenused in allergy diagnosis in a variety of indications, suchas inhalation allergies, allergies to insect venoms, foods,occupational allergens and various drugs. A large numberof reports on CAST diagnostic value, however, have beenanecdotal. A meta-analysis of validated and wellcontrolled studies encompasses 37 studies, 1614 patientsand 1145 controls. This should definitely establish thevalue of this diagnostic test, particularly in cases whereother in vitro or in vivo diagnostic tests are not reliable,such as food or drug allergies, as well as in non-IgE-mediated immediate hypersensitivity reactions.
However, a number of questions about the CASTdiagnostic assay are still open or have not beensystematically explored. This may explain, in additionto the practical limitations inherent to all allergy cellulartests, why CAST has not yet become a very widely usedassay worldwide, having gained broad acceptance insome countries but not in others.
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Prof. Dr. María L. Sanz
Department of Allergology and Clinical ImmunologyUniversity Clinic of NavarraApartado 420931080 Pamplona, [email protected]: 34 948 25 54 00Fax.: 34 948 29 65 00
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