We have monitored changes in basophil sensitivity as the logarithm of the concentration of allergen that elicits a half-maximal response, and blood basophil concentration, during the up dosing phase of SIT. We have found that there is a transient increase in basophil sensitivity after 3 weeks of SIT. This increase in sensitivity may reflect competing amounts of immunoglobulin on basophils (IgE) and free immunoglobulin in circulation (IgX, all classes of immunoglobulin with affinity for haptens on allergen). It coincides with the early decrease in basophil histamine release and increase in free IgG4 and IgA described recently .
During ultra rush procedures of SCIT with Japanese cedar  or birch or cat , maximal expression of CD203c as % activated cells on basophils  or MFI  after cross linking with allergen decreased with time. BAT could not predict results of a sting challenge at 0,1 or 1,0 ug venom . In a 5-day rush up dosing regimen, BAT at suboptimal concentrations appeared elevated after 5 days corresponding to the findings after 3 weeks in the present 16-week regimen (the significance of this had not been tested). Basophil activation was significantly reduced after six months and after three years . SCIT for birch or grass allergy significantly decreased CD-sens (analogous to LC50) tenfold , where the present results did not show a significant change. This may be because in the present experiment, BAT was done during the first 7 weeks assuming that all patients would complete up dosing by a semi rush protocol. Only 10 of 20 patients completed the semi-rush protocol. The protective component in plasma (that corresponds to Allergen Binding Activity (ABA)) increased by 1,04 LC50 units. This corresponds well to the tenfold change in ABA published recently .
Change in basophil activation measured as CD63 or CD203c activation at 0,1 ug/ml allergen did not vary significantly during the up dosing phase. A ratio of CD63 activation at 0,1 ug/ml/1 ug/ml > 92% has previously been shown to associate with severe side effects . In this study, 8 patients had a ratio > 92%. This did not associate with side effects (Table 1). In a study of modified rush up dosing of insect venom, the ratio of basophil activation at 0,1 ug/ml venom/1 ug/ml venom above 92% at baseline predicted severe side effects during up dosing . In the present study, 8 of 18 patients had a ratio of basophil activation predicting severe side effects. Neither others  nor we could confirm the usefulness of this ratio.
The up dosing scheme in the present study is much slower, and associated with very few side effects. This may explain that we do not find the decrease that was observed in the other studies, and why there were no side effects to associate with a high ratio of activation. The ratio of cell bound IgE to total free immunoglobulin may be the most significant effect parameter, which determines whether a basophil activation test becomes positive. The protective effect of SCIT in patient plasma (which we assume contains immunoglobulins competing for epitopes on allergen) had increased by >1 LC50 unit when comparing patients at baseline with patients just initiating maitenance therapy. Basophil activation has been shown to correlate well with clinical measures . Our study did not extend long enough to demonstrate an effect of treatment on basophil activation by flow cytometry, which has been shown by others . Inhibition of histamine release, the gold standard for basophil activity, was significantly different from baseline after 6 weeks of SCIT . Whilst single concentration analyses of basophil activation may not be sufficient to monitor effects of SCIT , we confirm here that this can be done with logarithmic series of dilutions of allergen.
CD-sens is the fist algorithm developed to measure basophil sensitivity in a basophil activation test . The allergen dose giving maximal activation is determined, and a line of regression is calculated through that point and the activation rates at the two preceding allergen concentrations. The half-maximal activation is determined from this interpolated line. CD-sens  was evaluated as a technique to calculate basophil sensitivity, but was not found to be useful in our hands because the allergen concentrations we used were too low to obtain a maximal activation as most of the curves we obtained were not bell shaped, and because our log dilutions were too widely spaced to obtain accurate CD-sens. LC50 considers more values than CD-sens, and may be less subjective. The values obtained with LC50 were larger than those obtained with CD-sens.
The present data gave us an opportunity to determine the optimal allergen concentration for diagnosing wasp allergy with a ROC curve to be 0,1 ug/ml final concentration in the reaction vessel with blood, which compares well with 0,5 ug/ml  and 0,5 - 0,05 ug/ml  final concentration of V vespula allergen in BAT published by others. The ROC curves for CD63 and CD203c and the threshold for positivity were almost identical for CD63 and CD203c, suggesting that the two markers measure similar aspects of basophil activation. Although Boumiza has disputed this , it has since been confirmed by a number of clinically oriented publications [24, 27, 28]. We have previously shown that choice of fluorochome and lysis procedure had favoured CD203c over CD63, illustrating the need to consider all aspects of an experiment . We have done the first Bland Altman analysis of CD63 and CD203c to show that in full blood there is no difference between the markers, whereas there is significantly less up regulation of CD203c than of CD63 on washed basophils. The effect appears not to be due to increased baseline expression of CD203c, as this was similar for both markers. Washing away plasma immunoglobulins increases the sensitivity of the test significantly, but masks the clinical relevance as the total response is compounded from the free immunoglobulin absorption of allergen and the cross linking of cell bound IgE.
The basophil numbers we obtained using CVA were within a recently published reference interval . Previously, we had shown that ingestion of allergen resulted in a 20% reduction of blood basophil concentration compared with placebo . Allergen injection, as a negative control, resulted in a marginal reduction in blood basophil numbers compared with controls. Changes in blood basophil concentration may thus be a marker for type I allergic response. As the difference in blood concentration of basophils increased with time from 30 min to 120 min, the optimal time frame for measuring blood basophil concentration as marker for a type I allergic reaction remains to be determined.
Discordance between in vivo, ex vivo and in vitro tests is a recurrent problem in allergen diagnosis, and some have suggested that basophil activation should be the arbitrating test if anamnesis and specific IgE do not support the same conclusion . We found a rate of discordance between patient history and in vitro diagnostic tests (3 patients of 18 with discordance between patient history and in vitro diagnostic tests) that was similar to others [23, 30]. The fact that basophil activation in full blood correlates with clinical symptoms and does reflect the response by cell-bound IgE in the context of competing immunoglobulins argues that it may occupy a special niche amongst diagnostic tests for allergy.