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J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269 © 2011 Esmon Publicidad
ORIGINAL ARTICLE
Serum Tryptase Level Is a Better
Predictor of Systemic Side Effects Than
Prostaglandin D2 Metabolites During Venom
Immunotherapy in Children
E Cichocka-Jarosz,1 M Sanak,2 A Szczeklik,2 P Brzyski,3 A Gielicz,2 JJ Pietrzyk1
1Chair of Pediatrics, Department of Pediatrics, Polish-American Children’s Hospital, Jagiellonian University
Medical College, Krakow, Poland
2Chair of Internal Medicine, Department of Internal Medicine, Jagiellonian University Medical College, Krakow,
Poland
3Chair of Epidemiology and Preventive Medicine, Department of Medical Sociology, Jagiellonian University
Medical College, Krakow, Poland
■ Abstract
Objectives: We performed a prospective study to analyze mast cell mediators as predictors of systemic adverse reactions during rush
venom-specifi c immunotherapy (VIT) in children.
Patients and Methods: Nineteen children aged 5-17 years received VIT with Venomenhal (HALAllergy). We analyzed serum tryptase (CAP,
Phadia), plasma prostaglandin (PG) D2 metabolites (9α,11ß-PGF2), and urine PGD2 metabolites (9α,11ß-PGF2, tetranor-PGD-M) using
gas chromatography mass spectrometry before and after the rush protocol.
Results: Three boys with high baseline serum tryptase values (>7.76 μg/L) (P<.001) and low 9α,11ß-PGF2 concentrations developed grade
III systemic adverse reactions during VIT. Baseline serum tryptase was lowest in children who had a Mueller grade II reaction (1.93 [0.36])
before VIT and highest in children with a Mueller grade III reaction (6.31 [4.80]) (P=.029). Repeated measures analysis of variance confi rmed
that, in children who developed systemic adverse reactions during VIT, serum tryptase was higher both before and after desensitization
and increased signifi cantly following the procedure. Analysis of PGD2 metabolites in the prediction of systemic adverse reactions during
VIT was inadequate (sensitivity 67% and specifi city 0.53%), whilst prediction based on serum tryptase was accurate.
Conclusions: In children with severe systemic adverse reactions to Hymenoptera sting, the evaluation of baseline tryptase levels should be a
standard procedure. Children with Apis mellifera venom allergy and baseline tryptase levels higher than 7.75 μg/L are at risk of anaphylaxis
during buildup. Lower baseline values of plasma and urinary PGD2 metabolite concentration in patients with systemic adverse reaction
during VIT suggest that prostaglandin catabolism is altered.
Key words: Rush venom immunotherapy. Children. Serum tryptase. 9α,11ß-PGF2. Tetranor-GD-M. PGD2 metabolites.
■ Resumen
Objetivos: Se realizó un estudio prospectivo para analizar los mediadores de los mastocitos como factores predictivos de reacciones adversas
sistémicas durante la inmunoterapia rápida específi ca con veneno en niños.
Pacientes y métodos: Diecinueve niños de entre 5 y 17 años de edad recibieron inmunoterapia con veneno con Venomenhal (HAL Allergy).
Se analizaron la triptasa sérica (CAP, Phadia), los metabolitos plasmáticos de la prostaglandina (PG) D2 (9α,11ß-PGF2) y los metabolitos
urinarios de la PGD2 (9α,11ß-PGF2, tetranor-PGD-M), utilizando cromatografía de gases y espectrometría de masas antes y después del
protocolo rápido.
Resultados: Durante la inmunoterapia con veneno, 3 niños con valores iniciales altos de triptasa sérica (>7,76 μg/l) (p<0,001) y
concentraciones bajas de 9α,11ß-PGF2 desarrollaron reacciones adversas sistémicas de grado III. Los niveles iniciales de triptasa sérica
fueron más bajos en los niños que, antes de la inmunoterapia con veneno, experimentaron una reacción de grado II en la escala de Mueller
(1,93 [0,36]), y más elevados en los niños con una reacción de grado III en la escala de Mueller (6,31 [4,80]) (p=0,029). Los análisis
Tryptase and PGD2 Metabolites During Venom Immunotherapy
© 2011 Esmon Publicidad J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269
261
Introduction
Severe systemic reaction to Hymenoptera sting is a
potentially life-threatening event. It is caused by a sudden
release of mediators derived from mast cells and basophils
upon exposure to venom allergens. Demonstration of a rapid
and transient increase in serum tryptase level (active mature
ß tryptase) during an allergic reaction refl ects massive mast
cell activation and confi rms the diagnosis of anaphylaxis [1].
Baseline serum mast cell tryptase concentration (inactive
α-/ ß-protryptases) refl ects a constitutive mast cell load or
activity and is considered to be a marker of mast cell clonal
disorders (mastocytosis) [2]. Elevated baseline serum tryptase
level has recently been shown to predict severe systemic reaction
both to Hymenoptera stings and during the buildup phase
of venom immunotherapy (VIT) in adults [3,4]. As baseline
tryptase level seems to increase continuously with age, more
severe anaphylactic reactions are observed in elderly people [5].
The prostaglandin (PG) D2 metabolites–9α,11ß-PGF2
and tetranor-PGD-M–reflect systemic PGD2 production
and are derived exclusively from mast cells and basophils.
These mediators are relatively stable and have proven useful
in monitoring asthmatic adults [6,7] and children [8,9]. They
have also been investigated in children with atopic eczema/
dermatitis using quantifi cation of urine 9α,11ß-PGF2 [10].
Data on 9α,11ß-PGF2 urinary excretion as a reliable marker
of endogenous production of proinflammatory PGD2 in
anaphylaxis are scant [11]. We present the preliminary results
of a similar approach in monitoring systemic adverse reactions
during VIT in children sensitized to Hymenoptera venom.
Quantification of eicosanoid production using gas
chromatography-negative ion chemical ionization-mass
spectrometry (GC-NICI-MS) is considered to be the gold
standard for reliable routine quantifi cation of eicosanoid
production in vivo [12,13]. Few studies monitor mast cell
mediators in children with Hymenoptera venom allergy.
Our objective was to assess the predictive value of mast
cell mediators (serum tryptase, plasma and urine 9α,11ß-PGF2,
and urine tetranor–PGD-M) in systemic adverse reactions
in children sensitized to Hymenoptera venom who were
prospectively recruited to undergo a rush VIT protocol.
de la varianza con determinaciones repetidas confi rmaron que, en los niños que desarrollaron reacciones adversas sistémicas durante
la inmunoterapia con veneno, los niveles de triptasa sérica fueron más elevados tanto antes como después de la desensibilización, y
aumentaron de forma signifi cativa tras el procedimiento. El análisis de los metabolitos de la PGD2 como factor predictivo de reacciones
adversas sistémicas durante la inmunoterapia con veneno resultó insufi ciente (sensibilidad del 67% y especifi cidad del 0,53%), mientras
que la predicción basada en la triptasa sérica resultó exacta.
Conclusiones: En niños con reacciones adversas sistémicas graves a la picadura de himenópteros, la evaluación de los niveles iniciales de
triptasa debería ser un procedimiento habitual. Los niños con alergia al veneno de abeja y niveles iniciales de triptasa superiores a 7,75 μg/l
presentan riesgo de anafi laxia durante la acumulación. Los valores iniciales más bajos de concentración de metabolitos plasmáticos y
urinarios de la PGD2 en pacientes con reacciones adversas sistémicas durante la inmunoterapia con veneno indican que el catabolismo
de las prostaglandinas está alterado.
Palabras clave: Inmunoterapia rápida con veneno. Niños. Triptasa sérica. 9α,11ß-PGF2. Tetranor-PGD-M. Metabolitos de la PGD2.
Patients and Methods
The study sample comprised 19 children (15 boys) aged
5-17 years (mean [SD], 10.6 [3.6] years) who underwent
VIT (10 to Apis mellifera venom, 9 to Vespula venom). The
inclusion criteria were systemic reaction to Hymenoptera
sting (Mueller grade II-IV) and confi rmed immunoglobulin
E(Ig)–-mediated allergy to venom. Three to six weeks after
fi eld systemic sting reaction, we performed skin prick tests
with Vespula species venom extract and Apis mellifera venom
extract (HALAllergy, The Netherlands) at a concentration
100 μg/mL, intradermal tests with updosing to the maximum
concentration of 1 μg/mL, and serum specifi c IgE (SSIgE)
determination (CAP System specific IgE FEIA, Phadia,
Uppsala, Sweden). The results were interpreted as described
elsewhere [1]. The clinical characteristics of the patients and
the results of the assays are presented in Table 1. Children
fulfi lling the inclusion criteria started an 8-day rush protocol
with incremental doses of venom (Venomenhal, HALAllergy)
(cumulative dose equal to 226.7 μg) (Table 2). Peripheral
venous blood and urine samples were taken twice in order to
estimate levels of mast cell mediators at baseline, ie, before
the fi rst dose of rush VIT (blood, morning in the fasting state;
urine, fi rst morning sample), and after the last injection of the
incremental dose (blood, after 5 minutes for 9α,11ß-PGF2
and 1 hour later for tryptase; urine, within 1-2 hours after the
last injection). Blood samples for tryptase were allowed to
clot, and serum was separated by centrifugation and stored at
–80ºC. Total α- and ß-proforms and mature ß tryptase were
measured using a fl uoroenzyme immunoassay based on the
CAP System (Phadia). The tryptase detection method had a
range of 1 to 200 μg/L, while normal values were considered
to be below 10 μg/L [14]. In the case of values greater than
10 μg/L, we performed duplicate measurements. According to
the manufacturer, the interassay variability for tryptase levels
between 1.0 and 100 μg/L is below 5%. Blood samples for
9α,11ß-PGF2 were immediately centrifuged at 3500 rpm for 10
minutes and 0.5 ng of internal deuterated standard PGF2α([2H4]
PGF2α) (CaymanChemicals, AnnArbor, Michigan, USA) was
added to 1 mL of plasma. Internal deuterated standard PGF2α
([2H4] PGF2α), was also added to 0.5 mL of urine to correct
J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269 © 2011 Esmon Publicidad
262 E Cichocka-Jarosz, et al
Table 1. Clinical Characteristics and Results of Assays With Specifi c Venom Allergy in Treated Patients
Patient number 1 2 3 4 5 6 7 8 9 10a 11 12 13 14 15b,c 16d 17 18 19
Gender Girl Boy Boy Boy Boy Boy Boy Boy Boy Boy Girl Boy Boy Boy Boy Girl Girl 1 1
Age, y 5 6 8 10 11 11 11 12 14 6 6 9 10 10 10 15 15 16 17
Systemic adverse reaction 1 1 1 1 1 1 1 1 1 2 1 2 2 1 1 1 1 1 1
Venom allergy (1, Vespula; 2, Apis mellifera) 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2
Mueller grade before VIT 4 3 4 4 3 3 4 2 4 3 4 3 3 2 4 4 2 4 2
Atopy (positive SPT result to inhalant allergens) Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Neg Neg Neg Neg Pos Pos Neg Neg Neg
Total IgE, kUA/L 46 2 126 72.10 80.70 157 308 81 495 80 49 262 489 207 424 374 147 243 202.38
Vespula venom sIgE, kUA/L 0.75 3.37 2.29 6.63 3.04 1.42 11.30 0.40 1.09 0.34 0.40 0.64 1.92 0 1.75 7.95 1.76 0.43 1.69
Honeybee venom sIgE, kUA/L 0 1.69 0 1.46 0 0 0 0 101 10.30 23.30 66.30 101 1.81 0.70 10.60 26 56.90 32.99
IDT Vespula venom concentration 1.0 μg/mL 10 11 9 7 7 7 9 7 5 0 0 0 2 7 0 0 0 5 0
IDT Apis mellifera venom concentration 1.0 μg/mL 0 0 0 0 5 0 4 0 10 0 0 9 8 5 0 3 9 9 7
(0.01 (0.1 (0.01
μg/mL) μg/mL) (μg/mL)
Baseline serum tryptase, μg/L 3.52 3.62 2.98 3.44 2.44 1 4.49 1.52 2.16 9.42 2.78 7.76 13.60 1.87 4.52 3.17 2.40 3.10 1.94
Serum tryptase after VIT 2.68 2.94 3.29 3.21 2.79 1.58 3.91 1.70 2.37 29.90 4.10 10.30 16.70 2.68 4.47 3.44 2.57 6.85 2.42
Baseline plasma 9α,11ß-PGF2 concentration, pg/mL 2.90 3.90 2.60 6.50 7.70 2 0.95 3.50 2.80 2.40 3.40 3.80 6.70 3 14.70 4 17 2 8.50
Plasma 9α.11ß-PGF2 concentration after VIT 23 7.50 4.30 4.80 2.50 4.10 0.51 6.10 2.30 9.70 16.50 11.80 24.90 5.20 30.90 6.20 4.50 7.30 7.80
Baseline urine 9α.11ß-PGF2 concentration,
ng/mg creatinine 0.81 0.63 0.50 1.39 5.90 0.72 1.13 0.80 0.60 1.10 0.50 0.20 0.53 0.40 0.60 0.63 0.90 0.30 0.40
Urinary 9α,11ß-PGF2 concentration after VIT 0.27 0.07 0.60 0.48 1 0.31 0.10 0.90 0.40 0.40 0.60 0.20 0.59 1 0.61 0.86 0.50 0.29 0.50
Baseline urine PGDM concentration, ng/mg creatinine 1.24 0.92 0.44 0.66 5.67 0.81 0.14 0.76 0.85 2.56 1.46 0.94 0.73 1.63 1.33 0.25 0.70 0.51 0.67
Urinary PGDM concentration after VIT 0.99 0.11 1.46 0.48 1.15 2.79 0.42 1.05 0.78 4.52 2.27 0.56 5.44 3.40 1.34 0.65 0.65 1.25 0.76
Abbreviations: IDT, intradermal test; Ig, immunoglobulin; PG, prostaglandin; SPT, skin prick test; VIT, venom immunotherapy.
aPolysensitization to inhalant allergens. Bronchial asthma, allergic rhinitis.
bPatient who could have received specifi c immunotherapy with both venoms but who was included in the category of patients treated against bee venom allergy, as his parameters were collected during Apis mellifera rush VIT,
which was completed before his treatment using Vespula venom.
cPositive SPT to Dermatophagoides farinae and Dermatophagoides pteronyssinus. Allergic rhinitis, episodic bronchial asthma.
dPositive SPT to Dermatophagoides farinae. Mild allergic rhinitis.
Tryptase and PGD2 Metabolites During Venom Immunotherapy
© 2011 Esmon Publicidad J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269
263
Table 2. Eight-Day Rush Protocol of Venom Immunotherapy Dose Increases
Day of VIT Venom Extract Daily Doses Cumulative
Concentration, μg/mL of Venom mL Daily Dose, μg
1 0.0001 0.1+0.2+0.4+0.8 0.00015
2 0.001 0.1+0.2+0.4+0.8 0.0015
3 0.01 0.1+0.2+0.4+0.8 0.015
4 0.1 0.1+0.2+0.4+0.8 0.15
5 1.0 0.1+0.2+0.4+0.8 1.5
6 10.0 0.1+0.2+0.4+0.8 15.0
7 100.00 0.1+0.2+0.3+0.4 100.00
8 100.00 0.5+0.6 110.00
Total cumulative dose 226.66665
for the loss of analyte during sample
preparation. All samples were stored
at –80º C and assayed within 1 month.
9α,11ß-PGF2 and tetranor-PGD-M
were measured using GC-NICIMS
(model 5896 series II; Hewlett
Packard, Palo Alto, California, USA)
as described elsewhere [6,15,16]. The
diagnostic ions were at m/z 569 and
m/z 573 for the internal standard of
9α,11ß-PGF2, and at m/z 489 and
m/z 495 for tetranor-PGD-M. The
detection limit was 1 pg/mL in plasma
samples and 0.5 ng/mg of creatinine
in urine samples.
Three patients were atopic
(positive skin prick tests Nexter/
Allergopharma with inhalant
allergens) (Table 1). On each VIT
day, patients were examined to rule
out any symptoms of infection. Stable
clinical condition and peak expiratory
fl ow over 80% of normal value were
verifi ed. None of the patients had
a history of recurrent urticaria or
any clinical symptoms of urticaria
pigmentosa. Renal function was
normal. No antihistamines, systemic
corticosteroids, or leukotriene
antagonists were administered during
VIT. No systemic adverse effects of
VIT were recorded.
Statistical Analysis
Results were described using
standard descriptive statistics (mean
[SD], range). Comparison within the
group of variables measured at 2 time
points was performed using the exact
Wilcoxon signed rank test. Variables
measured at the same time point were
contrasted between the groups using
the Mann-Whitney test or Kruskal-
Wallis test for more than 2 groups
when the grouping variable was
nominal or with a Jonckheere-Terpstra
test when the grouping variable was
ordinal. The strength of correlation
between variables measured on at
least ordinal level was estimated
using the Kendall τ-b coefficient.
Changes in mediators during VIT,
stratifi ed according to the occurrence
of systemic adverse effects, were
evaluated using univariate repeated
measurements analysis of variance
(ANOVA). Statistical significance
was set at P<.05.
Abbreviations: AR, allergic rhinitis; BA, bronchial asthma; Ig, immunoglobulin; PEF, peak expiratory
fl ow; PG, prostaglandin; SAR, systemic adverse reaction; SSIgE, serum specifi c IgE; VIT, venom
Table 3. Characteristics of Boys With Grade III Systemic Reaction During Honeybee Rush VIT
Patient 1 Patient 2 Patient 3
Dose which provoked SAR
reaction during VIT, μg 30 20 3
Age, y 10 12 6
Pretreatment Mueller grade III III III
Number of stings before reaction 10 2 0
Exposure to culprit insect High High Medium
Atopy presence No No Yes, AR,
mild chronic BA
Total IgE, kUA/L 262 489 80
Clinical symptoms of SAR/ Urticaria, Urticaria, Sneezing,
provoking dose, μg wheezing/ wheezing/ wheezing/
3 μg 20 μg 30 μg
SSIgE to Apis mellifera, kUA/L 6.3 101.0 10.3
SSIgE to Vespula, kUA/L 0.64 1.92 0.34
SS Apis mellifera IgE/t IgE ratio 0.253 0.207 0.129
Baseline tryptase, μg/L 7.76 13.60 9.42
Tryptase after rush VIT 10.30 16.70 29.90
Baseline plasma 9α,11ß-PGF2
concentration, pg/mL 3.80 6.70 2.40
Plasma 9α,11ß-PGF2 concentration
after rush VIT 11.80 24.90 9.70
Baseline urinary 9α,11ß-PGF2
concentration, ng/mg creatinine 0.20 0.53 1.10
Urine 9α,11ß-PGF2 concentration
after rush VIT 0.20 0.59 0.40
Baseline urinary PGDM
concentration 0.94 0.73 2.56
Urine PGDM after rush VIT,
ng/mg creatinine 0.56 5.44 4.52
PEF, % normal values 110 98 89
J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269 © 2011 Esmon Publicidad
264 E Cichocka-Jarosz, et al
Table 4. Comparison of Parameters Measured in 2 Detection Points for Children Allergic to Vespula Species and
Honeybee
Vespula Species Apis mellifera
Baseline serum tryptase
concentration, μg/L 2.80 (1.11) NS 5.06 (3.93) P=.007
Serum tryptase concentration
after rush VIT 2.72 (0.75) 8.34 (8.81)
Baseline plasma 9α,11ß-PGF2
concentration, pg/mL 3.99 (2.03) NS 6. 55 (5.31) P=.047
Plasma 9α,11ß-PGF2
concentration after rush VIT 6.83 (6.76) 12.48 (8.96)
Baseline urinary 9α,11ß-PGF2
concentration, ng/mg creatinine 1.39 (1.72) P=.021 0.56 (0.27) NS
Urinary 9α,11ß-PGF2
concentration after rush VIT 0.46 (0.33) 0.56 (0.24)
Baseline tetranor-PGDM
urinary concentration,
ng/mg creatinine 1.28 (1.68) NS 1.08 (0.68) P=.028
Tetranor-PGDM urinary
concentration after rush VIT 1.03 (0.78) 2.08 (1.78)
Abbreviations: NS, nonsignifi cant; PG, prostaglandin; VIT, venom immunotherapy.
The predictive value of PGD2 metabolites for systemic
adverse effects during VIT was estimated using a receiver operator
characteristics (ROC) curve for each PGD2 metabolite separately.
The best sensitivity-to-specifi city ratio was reported [17]. Positive
or negative predictive value, defi ned as percentages of correctly
classifi ed cases with and without adverse systemic reactions, were
computed. [18]. An area under the ROC curve (AUC) close to
0.5 means that prediction of a systemic adverse reaction using the
predictor is no better than a result due to chance.
Results
Clinical Findings
Grade III systemic adverse events were observed in 3
boys, 1 of whom was atopic (Table 3). The results of baseline
respiratory tests were normal, with no clinical symptoms of
asthma in the pretreatment physical examination. The patients
had not used ß-agonists within the 6 months before VIT. No
Table 5. Arithmetic Means of Analyzed Parameters Before and After Rush VIT With Regard to Occurrence of an SAR
Serum tryptase 9α, 11ß-PGF2 9α, 11ß-PGF2 PGDM urine
concentration plasma concentration, urine concentration, concentration,
μg/L pg/mLc ng/mg creatinine ng/mg creatinined
Before After Before After Before After Before After
Total 3.99 5.68 5.41 9.97 0.95 0.51 1.17 1.58
VIT and no SAR 2.81 3.19 5.63 8.87 1.01 0.53 1.13 1.22
VIT and SAR 10.26 18.97 4.30 15.47 0.61 0.40 1.41 3.51
Abbreviations: PG, prostaglandin; SAR, systemic adverse reaction; VIT, venom immunotherapy.
aDifference between children with and without SAR at baseline: P=.000
bDifference between children with and without SAR after rush VIT: P=.000
cDifference between total baseline and after VIT means: P=.032
dDifference between children with and without SAR after rush VIT: P=.009
systemic adverse events were observed during rush VIT in
the other children.
Baseline serum tryptase levels in children with no adverse
reactions during rush VIT were signifi cantly lower (P<.001)
than in children with adverse reactions (no higher than 4.52 μg/L).
No signifi cant differences were observed in baseline tryptase
values between children sensitized to Vespula species and
children sensitized to Apis mellifera (Table 4).
Differences in the baseline tryptase level according to
Mueller grade before VIT were signifi cant. The lowest serum
tryptase level was observed in children with a Mueller grade II
reaction (1.93 [0.36] μg/L), while the highest was in children
Tryptase and PGD2 Metabolites During Venom Immunotherapy
© 2011 Esmon Publicidad J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269
265
with a Mueller grade III reaction (6.31 [4.80] μg/L; P=.029).
Post hoc pairwise comparisons showed that the differences
between median baseline tryptase level of grade II vs III and
grade II vs IV were also signifi cant (P=.048 and P=.014,
respectively). After exclusion of the 3 children with grade
III systemic adverse reactions during VIT, baseline tryptase
concentration correlated positively with the Mueller grade
(P=.024), although only the difference between grade II and
grade IV retained its signifi cance.
Baseline values of urinary 9α,11ß-PGF2 were
signifi cantly lower in children with allergy to Vespula
venom (3.99 [2.03] ng/mg of creatinine) than in children
who were allergic to Apis mellifera venom (6.55 [5.31];
P=.023) (Table 4). After exclusion of the 3 children with
grade III adverse reactions during rush VIT, this difference
lost its signifi cance.
We observed a negative correlation between age and
baseline serum tryptase level (τ-b=–0.35; P=.044) and urinary
9α,11ß-PGF2 excretion (τ-b=–0.37; P=.031).
A negative correlation between SSIgE and baseline
urinary concentration of PGD2 metabolites was observed.
In children allergic to Vespula species, the correlation was
negative between SSIgE and tetranor-PGDM (τ-b=–0.49;
P=.006). Likewise, in children allergic to Apis mellifera, the
correlation was negative between SSIgE and 9α,11ß-PGF2
(τ-b=–0.38; P=.042).
Comparison of the Markers Measured Before and
After Rush VIT
Gender and atopy did not affect changes in mediators at the
2 assessment points. Table 4 summarizes the comparisons of
parameters between these samples separately for children allergic
to Vespula species and children allergic to Apis mellifera. In the
Apis mellifera–allergic group the markers increased following
rush VIT, except for urine 9α,11ß-PGF2. In children sensitized
to Vespula species, the 9α,11ß-PGF2 urine concentration was
higher at baseline and lower following VIT. Mean serum tryptase
and plasma 9α,11ß-PGF2 were signifi cantly higher in Apis
mellifera–allergic children than in Vespula-allergic children;
however, these differences disappeared when we excluded the
3 patients with systemic adverse reactions.
1.0
0.8
0.6
0.4
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0
Serum tryptase level
Sensitivity
1.0
0.8
0.6
0.4
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0
9α, 11ß-PGF2 Plasma concentration
Sensitivity
1.0
0.8
0.6
0.4
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0
Urine 9α, 11ß-PGF2 concentration
Sensitivity
1.0
0.8
0.6
0.4
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0
Tetranor-PGDM urine concentration
Sensitivity 1–Specifi city 1–Specifi city
1– Specifi city 1–Specifi city
Figure 1. Receiver operating characteristic curves for parameters predicting the risk of severe systemic reaction to rush venom immunotherapy.
J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269 © 2011 Esmon Publicidad
266 E Cichocka-Jarosz, et al
30.000
Before VIT After VIT
Change in serum tryptase level
8-day Venomenhal bee VIT
μg/L
25.000
20.000
15.000
10.000
5.000
0.000
40.000
Before VIT After VIT
Change in 9α, 11ß, PGF2 serum concentration
8-day Venomenhal bee VIT
pg/L
30.000
20.000
10.000
0.000
1.200
Before VIT After VIT
Change in 9α, 11ß-PGF2 urine concentration
8-day Venomenhal bee VIT
Creatinine, ng/mg
1.000
0.800
0.600
0.400
0.200
6.000
Before VIT After VIT
Change in PGDM urine concentration
8-day Venomenhal bee VIT
Creatinine, ng/mg
5.000
4.000
3.000
2.000
1.000
0.000
Figure 2. Individual excretion of serum tryptase, plasma 9α,11ß-PGF2, urine 9α,11ß-PGF2, and urine tetranor-PGD-M in children treated with specifi c
immunotherapy to Apis mellifera venom. VIT indicates venom immunotherapy.
Children With Systemic Adverse Reactions During
Buildup
Baseline serum tryptase levels in the children with systemic
adverse reactions during VIT were higher than in children
without reactions–the concentrations were higher than
7.76 μg/L in all 3. Table 5 summarizes the parameters analyzed
before and after rush VIT and stratifi es them according to the
presence of a systemic adverse reaction. The markers in 2 of
3 patients with reactions were lower than the mean level in
the group of children with no reactions during VIT. Serum
tryptase increased almost 2-fold during VIT and urinary
tetranor-PGD-M more than 2-fold. Plasma 9α,11ß-PGF2
increased almost 5-fold in children with reactions, although
this difference was not statistically signifi cant (Table 5).
Impact of VIT
Repeated measures ANOVA showed that the factors with
a signifi cant impact on serum tryptase level were sampling
points (ie, VIT treatment) (P=.001), occurrence of a systemic
adverse reaction (P=.000), and the interaction between the two
(P=.002). This parameter increased in both groups, but it was
signifi cantly greater in the children with an adverse reaction.
Analysis of plasma 9α,11ß-PGF2 concentration showed
only a signifi cant impact for VIT (P=.011), which caused
an increase in PG levels after immunization. No signifi cant
differences were observed between children who had an
adverse reaction and those who did not.
The results of the analysis for urine tetranor-PGD-M revealed
that both VIT (P=.006) and systemic adverse reaction (P=.019)
had a signifi cant impact on this metabolite. Immunization
increased excretion of tetranor-PGD-M in urine in both groups,
although the interaction between occurrence of a reaction and
sampling point was of borderline signifi cance (P=.052).
Urinary 9α,11ß-PGF2 excretion was not significantly
associated with VIT or adverse reactions.
Predicting the Properties of the Markers During
Buildup
Serum tryptase proved to be an excellent predictor
of systemic adverse reactions: all 3 children with
anaphylactic symptoms during VIT had increased baseline
levels (>7.76 μg/L).
Tryptase and PGD2 Metabolites During Venom Immunotherapy
© 2011 Esmon Publicidad J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269
267
5.000
Before VIT After VIT
Change in serum tryptase level
8-day Venomenhal wasp VIT
mcg/L
4.000
3.000
2.000
1.000
25.000
Before VIT After VIT
Change in 9α, 11ß-PGF2 serum concentration
8-day Venomenhal wasp VIT
mcg/L
20.000
15.000
10.000
5.000
0.000
6.000
Before VIT After VIT
Change in 9α, 11ß-PGF2 urine concentration
8-day Venomenhal wasp VIT
ng/mg creatinine
5.000
4.000
3.000
2.000
1.000
0.000
6.000
Before VIT After VIT
Change in PGDM urine concentration
8-day Venomenhal wasp VIT
ng/mg creatinine
5.000
4.000
3.000
2.000
1.000
0.000
Figure 3. Individual excretion of serum tryptase, plasma 9α,11ß-PGF2, urine 9α,11ß-PGF2, and urine PGD-M concentration in children treated with
specifi c immunotherapy to Vespula species. VIT indicates venom immunotherapy.
The other prostanoids were evaluated using ROC curves.
The sensitivity of both markers was at best 67%, and specifi city
did not exceed 53%. The highest specifi city was found for urine
9α,11ß-PGF2 concentration, with a cutoff of 0.62 ng/mg of
creatinine, assuming that lower values impose a higher risk of an
adverse reaction. The area under the curve for 9α,11ß-PGF2 was
60% (Figure 1), although this result was not signifi cant. Neither of
the prostanoid markers had a positive predictive value higher than
0.22; however, interestingly, the negative predictive value was not
less than 83%. Hence, neither of the studied PGD metabolites was
useful in predicting adverse reactions during VIT.
Individual levels of serum tryptase, plasma 9α,11ß-PGF2,
urinary 9α,11ß-PGF2, and urinary tetranor-PGD-M concentrations
in children treated with specifi c immunotherapy to Apis mellifera
and Vespula species venom are presented in Figures 2 and 3.
Discussion
We analyzed a profile of specific mast cell–derived
metabolites during rush VIT. To do so, we took into account
the kind of venom sensitization (Vespula species and Apis
mellifera) and systemic adverse effects during the buildup
phase of the protocol. No published studies have evaluated
serum tryptase in relation to plasma and urine concentrations
of PGD2 metabolites during rush VIT in children.
The main limitation of this study is its small population
and male predominance. This may refl ect a greater risk of
exposure to stings by boys, who take part in more outdoor
activities. More children were positive for venom SSIgE than
for intradermal tests. In a few nonatopic children presenting
high values of SSIgE, total IgE values were also elevated.
Three children were atopic. The frequency of atopy in the study
sample seemed comparable with that of the general population
[1]. Three children had a systemic adverse reaction (Mueller
grade III) to VIT, and 1 of these was diagnosed with atopic
asthma. This fi nding is relevant, as uncontrolled asthma is the
main risk factor of severe anaphylaxis in children [19]. We
did not fi nd any differences in the baseline values of mast cell
mediators according to gender and atopy; this observation is
consistent with published data [6,8]. The baseline laboratory
parameter that allowed us to identify children with systemic
J Investig Allergol Clin Immunol 2011; Vol. 21(4): 260-269 © 2011 Esmon Publicidad
E Cichocka-Jarosz, et al
adverse reactions was signifi cantly higher baseline serum
tryptase levels. These children also had lowered urine
9α,11ß-PGF2 values. Neither total IgE nor venom specifi c
IgE were discriminative. VIT had an impact on the parameters
analyzed in patients who had systemic reactions and in those
who did not. These changes in the levels of mediators lost their
signifi cance after children with systemic adverse reactions to
VIT were excluded. The change in urine 9α,11ß-PGF2 values
following VIT was puzzling and unexpected. Serum tryptase
proved to be an infallible predictor of adverse reactions: all 3
children with baseline serum tryptase exceeding 7.76 μg/L had
reactions. PGD2 metabolites were poor predictors of systemic
adverse reactions, and their positive predictive value was
particularly unsatisfactory.
In children with no systemic adverse reactions, differences
in levels of mast cell mediators did not change signifi cantly
following rush VIT. The treatment protocol seems safe, as it
allowed the patients to tolerate a dose equal to several stings by
Vespula species or more than 4 Apis mellifera stings within 8
days. All the patients with an adverse reaction during VIT had
signifi cantly higher baseline serum tryptase values, a fi nding
that is consistent with those of other authors [20]. Ruëff et al
[3,4] recently pointed out elevated baseline serum tryptase
level as a predictor of severe systemic reaction both to fi eld
sting and during the buildup phase of VIT. In our study, the
mean baseline tryptase level was 2.80 (1.11) μg/L for children
allergic to Vespula species and 5.06 (3.93) μg/L in children
allergic to Apis mellifera, while in children with systemic
adverse reactions it exceeded 7.75 μg/L. Compared with data
from a multicenter cohort study on predictors of anaphylactic
reaction in adults [2], patients who did not have an adverse
reaction in our study had baseline serum tryptase levels lower
than the reference value 5.84 (8.36) μg/L. We conclude that
a routine evaluation of mast cell mediators in children with
severe systemic reaction to Hymenoptera stings might help
in planning immunotherapy. The safety of rush VIT protocols
in high-risk patients has already been described [21,22]. In
high-risk patients, depot extracts should be considered during
the maintenance phase [23]. It is important to investigate and
control respiratory symptoms in asthmatic children before
VIT. Our results are consistent with those of other authors,
who recommend special monitoring of patients treated with
bee venom [24].
The quality of allergen-specifi c immunotherapy should be
monitored using national surveys [25]. Our model did not take
into account circadian variation in serum tryptase level or the
decline in serum tryptase level during long-term Hymenoptera
VIT reported elsewhere [26,27].
The mass spectrometry technique applied to measure
PGD2 metabolites in our study has the highest specifi city
and sensitivity among the available methods used to analyze
prostanoid compounds in biological matrices (plasma, urine,
exhaled breath concentrate) [6-9,11,12,16]. Published data
on monitoring PGD2 metabolites in patients with bronchial
asthma suggest this is a sensitive method for evaluation of
PGD2 biosynthesis following bronchial challenge in allergic
asthma [6,8,9]. Only 1 paper reports urine 9α,11ß-PGF2 levels
as a more useful marker of anaphylaxis than serum tryptase in
patients with a history of anaphylaxis that reoccurred during a
provocation test to identify specifi c allergens [11]. The novel
fi nding of our study was that the only laboratory parameters
that allowed us to identify children at risk of systemic adverse
reactions during VIT were higher baseline serum tryptase and
lower urine 9α,11ß-PGF2 levels. Even though VIT affected
biomarker levels regardless of whether an adverse reaction
occurred during VIT, these differences lost their signifi cance
after exclusion of children who experienced an adverse
reaction. The fi nding of decreased urine 9α,11ß-PGF2 levels
following VIT merits further study, as it contrasts with the
changes observed in other markers and could indicate common
patterns of PGD2 metabolism among children sensitized to
Hymenoptera venom. However, reference concentrations of
9α,11ß-PGF2 in plasma and urine are not currently available.
Our study provided conclusive evidence that rush VIT
is safe in children with low baseline serum tryptase levels.
Elevated serum tryptase level is a good predictor of severe
systemic reactions during VIT.
Conclusions
Although clonal mast cell disorders are rare in children,
evaluation of baseline serum tryptase levels should be a
standard procedure to ensure optimal prognosis, monitoring,
and administration of VIT following severe systemic reactions to
Hymenoptera sting. Children sensitized to Apis mellifera venom
and whose baseline serum tryptase exceeds 7.76 μg/L should be
carefully monitored for systemic adverse reactions during the
buildup phase of VIT. Likewise, in adults, there is a need for
validation of cutoff values for baseline serum tryptase levels in
much larger populations. This would help to identify children
with a higher risk of systemic adverse reactions during VIT.
Lower plasma and urine 9α,11ß-PGF2 concentrations
are also associated with a higher risk of systemic adverse
reactions during VIT. Since no reference values are available
for this metabolite and interindividual variation is substantial,
the hypothesis of underlying metabolic alterations should be
tested using much larger populations of children.
Acknowledgments
Supported by a research grant from the Polish Ministry
of Science and Highschool Education (registration number
N N407 254134). We declare no fi nancial relationship with
the biotechnology/pharmaceutical manufacturers mentioned
in this manuscript.
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Manuscript received April 23, 2010; accepted for
publication November 3, 2010.
Ewa Cichocka-Jarosz
265 Wielicka Str.
30-663 Krakow, Poland
E-mail: mijarosz@cyfronet.pl
269
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