J ALLERGY CLIN IMMUNOL
VOLUME 120, NUMBER 3
to in this article. The Yoshimura article2 is thus wrongly
cited. For years, we used flow cytometry in the diagnosis
of Hymenoptera venom allergy and follow-up of venom
immunotherapy (VIT). Although our analyses are re-
stricted to patients with systemic reactions (large local
reactions not being an indication for VIT 4,5) and we
have been using a whole blood assay, we were surprised
by some findings here. In our experience insufficient baso-
phil recovery is rare. Therefore we are surprised by a drop-
out of 14 of 35 patients. Although little information is
provided about the basophil isolation technique, it is
somewhat surprising that only a purity of 8% was reached.
Others,6 using an almost similar technique, report a baso-
phil purity of greater than 80%. It is well known that
improper isolation techniques could result in in vitro acti-
vation of basophils having higher basal CD63 expression.
Having performed lots of basophil activation tests in
patients before and at different time points during early
and prolonged VIT, we did not observe increased sponta-
neous expression of CD63 compared with that seen in
asymptomatic control individuals.7 In other words, our
data do not support the hypothesis of a primed phenotype
in patients with venom allergy.
The authors state that the allergen doses applied in
basophil activation tests rest on experience with mediator
release tests. They suggest that basophil activation tests
could benefit from concentrations more relevant to those
achieved after in vivo stings. However, the authors failed
to identify some relevant publications. Several studies on
flow-assisted venom allergy diagnosis with dose-finding
experiments between patients and control subjects have
been published.7-9 One should also be aware that the
published stimulation concentrations are ‘‘working con-
centrations’’ and frequently do not reflect the final concen-
trations in the aliquots.
Finally, the authors state that allergen sensing by
basophils, as demonstrated by CD63 expression, persists
despite VIT. However, in the cited article,9 according to
their dose-finding experiments, the authors applied maxi-
mal stimulation of the basophils before sting challenge.
We demonstrated venom-induced CD63 upregulation
to significantly decrease during VIT, provided the cells
are stimulated submaximally.7 The final concentration
of venom in the aliquots to demonstrate this effect was
0.005 mg/mL, a concentration considered to be clinically
relevant.1
Didier G. Ebo, MD, PhD
Chris H. Bridts, MLT
Evelyne Dombrecht, PhD
Luc S. De Clerck, MD, PhD
Wim J. Stevens, MD, PhD
From the Department of Immunology, University Antwerp, Antwerp, Belgium.
E-mail: immuno@ua.ac.be.
Disclosure of potential conflict of interest: The authors have declared that they
have no conflict of interest.
REFERENCES
1. Gober LM, Eckman JA, Sterba PM, Vasagar K, Schroeder JT, Golden
DB, et al. Expression of activation markers on basophils in a controlled
Correspondence 727
model of anaphylaxis. J Allergy Clin Immunol 2007 [Epub ahead of
print].
2. Yoshimura C, Yamaguchi M, Iikura M, Izumi S, Kudo K, Nagase H, et al.
Activation markers of human basophils: CD69 expression is strongly and
preferentially induced by IL-3. J Allergy Clin Immunol 2002;109:817-23.
3. Buhring HJ, Streble A, Valent P. The basophil-specific ectoenzyme
E-NPP3 (CD203c) as a marker for cell activation and allergy diagnosis.
Int Arch Allergy Immunol 2004;133:317-29.
4. Bonifazi F, Jutel M, Bilo BM, Birnbaum J, Muller U. Prevention and
treatment of hymenoptera venom allergy: guidelines for clinical practice.
Allergy 2005;60:1459-70.
5. Moffitt JE, Golden DB, Reisman RE, Lee R, Nicklas R, Freeman T, et al.
Stinging insect hypersensitivity: a practice parameter update. J Allergy
Clin Immunol 2004;114:869-86.
6. MacGlashan DW Jr. Graded changes in the response of individual human
basophils to stimulation: distributional behavior of events temporally
coincident with degranulation. J Leukoc Biol 1995;58:177-88.
7. Ebo DG, Hagendorens MM, Schuerwegh AJ, Beirens LM, Bridts CH,
De Clerck LS, et al. Flow-assisted quantification of in vitro activated
basophils in the diagnosis of wasp venom allergy and follow-up of
wasp venom immunotherapy. Cytometry B Clin Cytom 2007;72:196-203.
8. Sturm GJ, Bohm E, Trummer M, Weiglhofer I, Heinemann A, Aberer W.
The CD63 basophil activation test in Hymenoptera venom allergy: a
prospective study. Allergy 2004;59:1110-7.
9. Erdmann SM, Sachs B, Kwiecien R, Moll-Slodowy S, Sauer I, Merk HF.
The basophil activation test in wasp venom allergy: sensitivity, specificity
and monitoring specific immunotherapy. Allergy 2004;59:1102-9.
Available online June 22, 2007.
doi:10.1016/j.jaci.2007.05.013
Reply
To the Editor:
We would like to address the comments cited in the
correspondence from Ebo et al1 in regard to our article.2
In regard to the effect of CD63 by IL-3, the Yoshimura
article3 was cited incorrectly, because they demonstrated
that CD63 is not significantly mobilized on basophils by
IL-3 alone but rather only with anti-IgE. However, recent
studies have shown increased spontaneous expression of
CD63 on the basophils of some atopic individuals after
preincubation with IL-3 or enhancement of allergen- or
anti-FceRI–induced CD63 expression.4,5 This latter find-
ing should be further explored because the basophil activa-
tion test might involve preincubating basophils with IL-3.
In regard to the comment on our basophil recovery,
patient blood samples were of a small volume (15 mL) and
isolated by using a Percoll-based separation technique.
We specifically avoided whole blood assays given our past
experience showing that this technique leads to higher
basal values of activation marker expression compared
with Percoll-based isolation because of the need for red
cell lysis.6 Dr Ebo referenced a basophil purity of 80%, but
this cited work, although using a Percoll-based separation
technique, also used additional purification by means of
elutriation, thus increasing basophil purity significantly.7
The basophil isolation procedure described in our article
uses a Percoll-based density gradient centrifugation tech-
nique that is well-described in the literature, with a re-
ported basophil purity of 2% to 23%.8,9
The authors also referred to our discussion on allergen
dose selection in basophil activation tests. Allergen dose
selection for in vitro stimulation is not always within the
728 Correspondence
range experienced by a field sting and varies between stud-
ies using the basophil activation test. Also, the amount of
clinically relevant venom might vary between different
Hymenoptera species and is unknown. We found that in
intentional sting challenge, those subjects with a history
of systemic reactions on multiple years of venom immuno-
therapy still had an increase in basophil CD63 values
in vivo after the sting. We did not perform graded
in vivo sting challenges to determine the CD63 response
to submaximal doses of venom because this would not
be feasible with a live sting challenge.
Laura M. Gober, MD
Sarbjit S. Saini, MD
From the Department of Medicine, Division of Allergy and Clinical Immu-
nology, The Johns Hopkins University School of Medicine, Baltimore, Md.
E-mail: ssaini@jhmi.edu.
Disclosure of potential conflict of interest: The authors have declared that they
have no conflict of interest.
REFERENCES
1. Ebo DG, Bridts CH, Dombrecht EJ, De Clerck LS, Stevens WJ. Expres-
sion of activation markers on basophils in a controlled model of anaphy-
laxis: general, methodological and clinical issues. J Allergy Clin Immunol
2007;120:726-7.
2. Gober LM, Eckman JA, Sterba PM, Vasagar K, Schroeder JT, Golden
DB, et al. Expression of activation markers on basophils in a controlled
model of anaphylaxis. J Allergy Clin Immunol 2007;119:1181-8.
3. Yoshimura C, Yamaguchi M, Iikura M, Izumi S, Kudo K, Nagase H, et al.
Activation markers of human basophils: CD69 expression is strongly and
preferentially induced by IL-3. J Allergy Clin Immunol 2002;109:817-23.
4. Cozon G, Ferrandiz J, Peyramond D, Brunet J. Detection of activated
basophils using flow cytometry for diagnosis in atopic patients. Allergol
Immunopathol 1999;27:182-7.
5. Ocmant A, Peignois Y, Mulier S, Hanssens L, Michils A, Schandene L.
Flow cytometry for basophil activation markers: the measurement of
CD203c up-regulation is as reliable as CD63 expression in the diagnosis
of cat allergy. J Immunol Methods 2007;320:40-8.
6. Vasagar K, Vonakis BM, Gober LM, Viksman A, Gibbons SP Jr, Saini
SS. Evidence of in vivo basophil activation in chronic idiopathic urticaria.
Clin Exp Allergy 2006;36:770-6.
7. MacGlashan DW Jr. Graded changes in the response of individual human
basophils to stimulation: distributional behavior of events temporally
coincident with degranulation. J Leukoc Biol 1995;58:177-88.
8. Saini SS, Richardson JJ, Wofsy C, Lavens-Phillips S, Bochner BS,
Macglashan DW Jr. Expression and modulation of FceRIa and FceRIb
in human blood basophils. J Allergy Clin Immunol 2001;107:832-41.
9. Warner JA, Reshef A, MacGlashan DW Jr. A rapid Percoll technique for the
purification of human basophils. J Immunol Methods 1987;105:107-10.
Available online June 22, 2007.
doi:10.1016/j.jaci.2007.05.006
Measuring asthma control is not just relevant
for clinical studies
To the Editor:
In the March 2007 issue of the Journal, Apter1 discusses
the studies about risk factors for asthma and its course and
management that were published in 2006 and are relevant
to clinical practice. The article covers a wide range of cur-
rent asthma topics in a condensed way. In the paragraph on
measuring asthma control, Apter1 notes that asthma con-
trol is an important variable in clinical studies. We fully
agree with this statement but would like to add that asthma
J ALLERGY CLIN IMMUNOL
SEPTEMBER 2007
control is not just an important variable in clinical studies
but also highly relevant for daily clinical practice. Seventy
percent or more of all patients have poorly controlled
asthma, and the majority of these patients do not realize
this.2,3 Therefore, to improve asthma control in our
patients, an objective tool to identify and quantify sub-
optimal asthma control would be of great help.
We also agree with the author that so-called biomarkers
to assess asthma control are too expensive and complex
for routine clinical application. Apter1 refers to the
Asthma Control Test4—a reliable self-report tool to mea-
sure asthma control—that has been validated for patients
with asthma with mild symptoms treated by secondary
care asthma specialists. However, this tool has not yet
been validated for detecting poorly controlled patients
among all patients with asthma. Because most patients
with asthma are diagnosed and treated in primary care,
especially in primary care a tool to identify poor asthma
control would be very helpful. For this situation, the
Asthma Control Questionnaire,5,6 which has been exten-
sively validated, may give primary care physicians a
good starting point to detect actively patients with uncon-
trolled asthma symptoms in their practice7 and improve
their level of asthma symptom control. Asthma control
is not only an important clinical study outcome; detecting
(and subsequently improving) poor asthma control in
daily life practice can actually lower the burden of the dis-
ease in the asthma population and make a difference for at
least some of our patients with asthma.
Lotte van den Nieuwenhof, MD
Patrick J. P. Poels, MD
Tjard R. J. Schermer, PhD
From the Department of Family Medicine, Radboud University Nijmegen
Medical Centre, The Netherlands. E-mail: L.vandennieuwenhof@hag.umcn.nl.
Disclosure of potential conflict of interest: T. R. J. Schermer is on the
speakers’ bureau for the International Primary Care Respiratory Group.
The rest of the authors have declared that they have no conflict of interest.
REFERENCES
1. Apter AJ. Advances in adult asthma 2006: its risk factors, course, and
management. J Allergy Clin Immunol 2007;119:563-6.
2. Rabe KF, Vermeire PA, Soriano JB, Maier WC. Clinical management of
asthma in 1999: the Asthma Insights and Reality in Europe (AIRE) study.
Eur Respir J 2000;16:802-7.
3. Lai CK, De Guia TS, Kim YY, Kuo SH, Mukhopadhyay A, Soriano JB,
et al. Asthma control in the Asia-Pacific region: the Asthma Insights and
Reality in Asia-Pacific Study. J Allergy Clin Immunol 2003;111:263-8.
4. Nathan RA, Sorkness CA, Kosinski M, Schatz M, Li JT, Marcus P, et al.
Development of the asthma control test: a survey for assessing asthma
control. J Allergy Clin Immunol 2004;113:59-65.
5. Juniper EF, O’Byrne PM, Guyatt GH, Ferrie PJ, King DR. Development
and validation of a questionnaire to measure asthma control. Eur Respir J
1999;14:902-7.
6. Juniper EF, Bousquet J, Abetz L, Bateman ED. Identifying ‘‘well-con-
trolled’’ and ‘‘not well-controlled’’ asthma using the Asthma Control
Questionnaire. Respir Med 2006;100:616-21.
7. Nieuwenhof van den L, Schermer T, Eysink P, Halet E, van WC, Bindels
P, et al. Can the Asthma Control Questionnaire be used to differentiate
between patients with controlled and uncontrolled asthma symptoms?
a pilot study. Fam Pract 2006;23:674-81.
Available online July 5, 2007.
doi:10.1016/j.jaci.2007.05.007