SEARCH

SEARCH BY CITATION

Keywords:

  • basophil;
  • Basotest®;
  • grass pollen;
  • monosensitization

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background: Basotest® is a new basophil-activation test based upon the expression of CD63 (gp53) in the presence of allergens. It is an effective diagnostic test for pollen-allergic patients. However, it is not known whether Basotest results differ during the pollen season.

Methods: We examined the activation of basophils by Basotest in 13 patients sensitized only to grass pollen, before and during the pollen season, in order to assess whether Basotest could be used as a diagnostic test during the pollen season. Dose-response curves with 10-fold increasing concentrations of timothy grass pollen (10−4 to 100 AU) were carried out.

Results: Basophils were not activated spontaneously during the pollen season since the CD63 expression was below detectable levels before in vitro cell activation. A decreased percentage of activated basophils at the peak of activation was found in comparing the pre- and in-season tests, but all patients had a positive test. When basophil activation was at its peak, the allergen concentration was similar during the two periods. Moreover, the median area under the curve was significantly (P<0.02) reduced during the season as compared to before the season.

Conclusions: Basotest can therefore be used as a diagnostic test during the pollen season, but the allergen exposure needs to be characterized if quantitative studies are performed.

Pollen allergy is very common and patients can be classified according to their sensitivities to allergens into mono- and polysensitized groups (1). Patients allergic to a single allergen (monosensitized) differ from those allergic to many allergen species (polysensitized), since the former have lower total serum IgE levels (2), and their peripheral blood mononuclear cells produce lower levels of IL-4 and IgE in vitro (3). Moreover, patients allergic only to cypress pollen may present an IgE-immune response similar to that found in nonallergic individuals (4, 5). During the pollen season, numerous changes have been observed. In the monosensitized group, IL-4 levels increase whereas no variation in IL-4 or IgE release was observed in the polysensitized group (6).

Basotest® (Becton Dickinson, BD, Pont de Claix, France) is a new basophil-activation test based upon the expression of CD63 (gp53) in the presence of allergens or nonspecific stimuli (7, 8). In this test, CD63 is measured by cytofluorometry. Basotest was found to be an effective diagnostic test in patients allergic to cypress pollen. It was more sensitive and specific than the measurement of serum specific IgE by the CAP® System (9).

During allergen exposure, basophils can be activated (10). Such an in vivo activation has been found in allergy to Hymenoptera venom (11), but it is not known whether the Basotest results vary according to the pollen exposure.

In the present study, we examined the activation of basophils with Basotest before and during the pollen season in patients sensitized only to grass pollen. The main objective of the study was to assess whether Basotest could be used during the pollen season as a diagnostic test.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients

Thirteen allergic patients (10 men), ranging in age from 20 to 50 years (mean±SD: 30.4±11.2 years), volunteered to enter the study after informed consent. The study was approved by the ethics committee of Montpellier University (France). All patients were allergic and selected according to a previous study (2) by the following criteria:

1) a suggestive history of seasonal allergic rhinitis

2) positive skin prick tests to orchard grass pollen, but no other allergen sensitization (Stallergènes, Antony, France)

3) the presence of a single band by IgE immunoprint to orchard grass pollen, as previously observed in patients sensitized only to grass pollen (2)

4) the presence of specific IgE to orchard and timothy grass pollen (Phadebas CAP System, Pharmacia-Upjohn, Uppsala, Sweden).

None of the patients had received any form of specific immunotherapy, and none had been treated by any form of corticosteroids for the previous 3 months, as these drugs might have had some immunomodulatory effects.

Basotest

We used Basotest for the quantitative determination of the in vitro basophil activation to grass pollen. The description of the technique has recently been published in detail (9). However, in the present study, we made a slight modification since we established the dose-response curve of the Basotest activation with 10-fold decreasing concentrations (100 to 10−4 AU) of the extract provided by the manufacturer (Greer Laboratories, Lenoir, NC, USA). Samples were incubated for 20 min at 4°C with 20 µl of antibody PE-anti IgE and anti-FITC-gp53 (Becton-Dickinson, BD, Pont de Claix, France). Erythrocytes were subsequently removed by the addition of 2 ml of lysing solution (BD). Cells were washed twice with PBS solution, resuspended in 200 µl of PBS solution, and then analyzed within 1 h by cytofluorometry (FASCalibur, BD). The basophil population was gated by the expression of PE anti-IgE. The expression of gp53 was analyzed on this gated cell population. The data acquisition was generally carried out on 1000 basophils, and we excluded the studies of the samples in which the acquisition had been performed on fewer than 200 cells. Results were given as the percentage of basophils expressing CD63. We analyzed the percentage of cells activated at the peak of the dose-response curve, the percentage of cells activated with the highest allergen concentration, and the area under the curve (AUC). A percentage of CD63 expression of 15% was considered a positive basophil degranulation according to the package insert and to our previous study (9).

The specificity of Basotest was confirmed with a nonsensitizing allergen (house-dust mite), and none of the patients had a basophil activation over 15% (2.0–6.5%).

We also studied the reproducibility of Basotest carried out twice in the same patients. There was no significant difference between the tests, confirming our previous study in which we observed a variation coefficient of 5.4% (9).

Six nonallergic subjects were tested during allergen exposure, and there was no basophil activation (2.1–3.5%).

Design of the study

Symptom scores were measured on diary cards assessing symptoms on a 0–3 scale, and patients were tested on two occasions; firstly, before the pollen season in asymptomatic patients, and, secondly, during the pollen season when patients had had at least a week of moderate to severe symptoms. Basotest was carried out on both occasions by the same technique.

Statistical analysis of the data

With the exception of demographic characteristics, in which means and SD were used, results were expressed in medians and 25–75th percentiles. Nonparametric tests were carried out. Intergroup analyses were performed with the Mann–Whitney U test. The intragroup analyses were performed with the Wilcoxon W test.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

All grass-pollen-allergic patients had a significant activation of basophils after allergen challenge, and, on three occasions only, the number of tested cells was under 200. Thus, all dose-response curves could be analyzed.

Basophils were not activated spontaneously during the pollen season since CD63 was always undetectable (2.7%, 1.7–4.2%) before in vitro cell activation.

When comparing the pre- and in-season tests, we found a significant difference (P=0.01) in the median percentage of activated basophils at the peak of activation (85.4% [77.2–92.5] vs 62.2% [58.0–72.8]) (pre- and in-season, respectively). The median allergen concentration for the peak of basophil activation was similar (1% [0.1–10] vs 1% [0.1–3.2]). There was a significant difference (P=0.0005) in the median percentage of activated basophils at the maximal allergen concentration used (77.1% [51.1–82.3] vs 52.0% [41.7–73.2]) (pre- and in-season, respectively). However, all patients had a basophil activation over 15% before and during the pollen season with this allergen concentration, which is recommended by the manufacturer (Fig. 2). Moreover, there was a significant decrease (P<0.02, Wilcoxon W test) in the median AUC during the pollen season (6.3cm2 [5.4–8.6]) compared to the area observed before the pollen season (9.6cm2 [7.6–13.4]) (Figs. 1 and 3).

image

Figure 2. Percentage of CD63+ cells observed after stimulation by grass-pollen allergen (100 µl) before and during pollen season. Individual data with median and 25–75th percentiles of population at each pollen season. Comparison between seasons was by Wilcoxon test.

Download figure to PowerPoint

image

Figure 1. Dose-response CD63 expression of basophils stimulated with 10-fold increasing concentrations of grass-pollen allergen before and during pollen season. Results are given in percentage of basophils expressing CD63.

Download figure to PowerPoint

image

Figure 3. Variation of AUC of dose-response curve of basophils before and during pollen season. Individual data with median and 25–75th percentiles of population at each pollen season. Comparison between seasons was by Wilcoxon test.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In the present study, we showed that although in vitro basophil activation decreases during the pollen season, Basotest is always positive and can be performed either during or outside the pollen season for diagnostic purposes. However, if quantitative studies are carried out, the time of the study may be critical, since the in vitro activation of the cells appears to be reduced during the pollen season (peak of activation of basophils and AUC).

The methods used in the present study have been validated, and we studied only very well-characterized patients monosensitized to grass pollens in order to avoid any spontaneous activation of basophils by other allergens. However, in the present study, we observed that spontaneous activation of basophils did not occur during the pollen season. The monosensitization of the patients was studied by skin tests. Moreover, the orchard-grass-pollen IgE immunoprint analysis exhibited only one band of 30 kDa, confirming previous data on monosensitized patients (2).

We have previously shown the reproducibility of Basotest, and this was confirmed in the present study. The results reported in the present study can therefore be analyzed (9).

The present study shows that Basotest can be used in the diagnosis of allergy to grass pollen since all patients included had a positive test. However, it is not usually necessary to use this test to make that diagnosis possible.

The overall in vitro activation of basophils was reduced during the pollen season since the AUC and the maximal level of activation were significantly reduced by comparison with preseason values. These data suggest that if precise studies are carried out on basophil activation they should take the allergen exposure of the patients into consideration. These findings may be due to a priming of cells, as has been suggested by the increased histamine levels in the serum of allergic patients (12, 13). However, further mechanistic studies are needed to clarify this phenomenon.

The aim of the study was to assess whether Basotest could be used as a diagnostic test during allergen exposure. The results show unequivocally that all patients who had a positive Basotest the pollen season also had a positive test during the season. However, as shown in Fig. 2, some patients had a reduction of AUC of over 50%. Thus, Basotest can be used as a diagnostic test, but if the aim of the study is to assess the quantitative activation of basophils, the time of the study may be critical, since differences can be observed. Moreover, the present study does not exclude the results of Basotest immediately or soon after anaphylactic reactions, in which the activation of basophils is probably more pronounced than in pollen allergy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was funded by INSERM and Stallergènes Laboratory. S.K. received a French government grant.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • 1
    Bousquet J, Cour P, Guerin B, Michel FB. Allergy in the Mediterranean area. I. Pollen counts and pollinosis of Montpellier. Clin Allergy 1984;14:249258.
  • 2
    Bousquet J, Hejjaoui A, Becker WM, et al. Clinical and immunologic reactivity of patients allergic to grass pollens and to multiple pollen species. I. Clinical and immunologic characteristics. J Allergy Clin Immunol 1991;87:737746.
  • 3
    Pene J, Rivier A, Lagier B, Becker WM, Michel FB, Bousquet J. Differences in IL-4 release by PBMC are related with heterogeneity of atopy. Immunology 1994;81:5864.
  • 4
    Reid MJ, Schwietz LA, Whisman BA, Moss RB. Mountain cedar pollinosis: can it occur in non-atopics? N Engl Reg Allergy Proc 1988;9:225232.
  • 5
    Bousquet J, Knani J, Hejjaoui A, et al. Heterogeneity of atopy. I. Clinical and immunologic characteristics of patients allergic to cypress pollen. Allergy 1993;48:183188.
  • 6
    Lagier B, Pons N, Rivier A, et al. Seasonal variations of interleukin-4 and interferon-gamma release by peripheral blood mononuclear cells from atopic subjects stimulated by polyclonal activators. J Allergy Clin Immunol 1995;96(6 Pt 1):932940.
  • 7
    Knol EF, Mul FP, Jansen H, Calafat J, Roos D. Monitoring human basophil activation via CD63 monoclonal antibody 435. J Allergy Clin Immunol 1991;88(3 Pt 1):328338.
  • 8
    Knol EF, Koenderman L, Mul FP, Verhoeven AJ, Roos D. Differential activation of human basophils by anti-IgE and formyl-methionyl-leucyl-phenylalanine. Indications for protein kinase C-dependent and -independent activation pathways. Eur J Immunol 1991;21:881885.
  • 9
    Paris-Köehler A, Demoly P, Persi L, Bousquet J, Arnoux B. In vitro diagnosis of cypress pollen allergy using cytofluorometric analysis of basophils (Basotest). J Allergy Clin Immunol 2000;105:339345.
  • 10
    Wilson SJ, Lau L, Howarth PH. Inflammatory mediators in naturally occurring rhinitis. Clin Exp Allergy 1998;28:220227.
  • 11
    Eberlein-Konig B, Ullmann S, Thomas P, Przybilla B. Tryptase and histamine release due to a sting challenge in bee venom allergic patients treated successfully or unsuccessfully with hyposensitization. Clin Exp Allergy 1995;25:704712.
  • 12
    Casolaro V, Spadaro G, Marone G. Human basophil releasability. VI. Changes in basophil releasability in patients with allergic rhinitis or bronchial asthma. Am Rev Respir Dis 1990;142:11081111.
  • 13
    Busse WW, Swenson CA, Sharpe G, Koschat M. Enhanced basophil histamine release to concanavalin A in allergic rhinitis. J Allergy Clin Immunol 1986;78(1 Pt 1):9097.