Analysis of in vitro-activated patient basophils
Currently, flow cytometric analysis of in vitro-activated patient basophils using CD63 and CD203c proved reliable for the diagnosis of different IgE-mediated allergies including classical inhalant allergens (pollen, house dust mite), primary and secondary food allergies, natural rubber latex allergy, hymenoptera venom allergy, and drug allergies (Table 2). In addition, the technique allowed component-resolved diagnosis of pollinosis (35) and hymenoptera venom allergy (34). Moreover, flow cytometric analysis of in vitro-activated basophils was repeatedly demonstrated to constitute a valuable instrument to assess and to compare the (residual) allergenicity of natural allergen extracts, chemically modified and recombinant allergens (4, 35, 37, 38, 62–64). Finally, most recently, flow-assisted analysis of in vitro-activated basophils has been applied to assess passive IgE-sensitization by blood transfusion (65).
Table 2. Flow cytometric analysis of in vitro allergen-activated basophils: sensitivity and specificity data
|House dust mite (HDM)||H + IgE and/or ST||56–78|| 91–100||20||(19)|
|Dactylis glomerata||H + IgE and/or ST||73–100||100|
|Cypress pollen||H + ST + PT||91||100||75||(48)|
|HDM and Lolium perenne||H + IgE + ST||93|| 98||128||(20)|
|Latex||H + IgE + ST||93|| 92||102||(23)|
|Latex||H + ST||93||100||73||(53)|
|Latex||H + IgE and/or ST||80|| 97||79||(74)|
|Wasp venom||H||92|| 80||70||(49)|
|Wasp venom||H||85|| 83||87||(58)|
|Bee venom||H||91|| 90|
|β-lactam||H + ST||50|| 93||88||(25)|
|β-lactam||H ± ST ± IgE ± PT||49|| 91||110||(84)|
|Metamizol||H ± PT||42||100||55||(85)|
|Aspirin and NSAID||H ± PT||15–55|| 74–100||90||(86)|
|Neuromuscular blocking agent||H||64|| 93||50||(51)|
|Neuromuscular blocking agent||H ± ST||54||100||60||(24)|
|Neuromuscular blocking agent||H||79||100||24||(27)|
|Carrot–celery–hazelnut||H (OAS)||85–90|| 80–90|| ||(81)|
|Apple (Mal d 1)||H (OAS)||75|| 64||54||(50)|
|Celery (Api g 1)||65|| 86|
|Carrot (Dau c 1)||75|| 82|
|Serum of patients with CIU||H + ASST||20|| 70||65||(102)|
The efficacy of flow cytometry particularly in terms of sensitivity is highly dependent on the allergen type investigated. For proteins (pollen, house dust mite, latex, venoms, foods) the picture is globally comparable and the efficacy might increase with the use of recombinants. For drugs, however, the much lower observed sensitivity of the test probably reflects a weaker allergenicity of these molecules. In fact, in order to elicit the IgE receptor aggregation pathway, allergens must be, at least divalent (66). Results obtained with the RBL-2H3 cell line (basophilic leukaemia cell line) have shown that high-order oligomers (trivalent, tetravalent, etc.) may induce a stronger signal leading to an increase of the IgE receptor clustering (67). Other factors are also involved as the lifetime of the clustering which must exceed 100 s (68) and the number of receptors clustered (69). The earlier steps may be also reversible (70). All these factors and the nature of the microenvironment may lead to selective basophil responses.
Inhalant allergens. Several studies have evaluated application of flow cytometry in the diagnosis of pollinosis (19–21, 35, 48). Overall, flow cytometric quantification of CD63 and/or CD203c on peripheral blood basophils exposed to natural and/or recombinant pollen allergens demonstrated a highly reliable approach to diagnose pollen allergy. In the study by Hauswirth et al. (35) a significant correlation between CD63 and CD203c expression (R = 0.76) was observed.
Two of the above-mentioned studies also investigated flow cytometric analysis of in vitro-activated basophils in the diagnosis of house dust mite allergy (19, 20). The overall sensitivity and specificity of flow-assisted diagnosis in classical inhalant allergy equalled or exceeded 90%.
Latex. Application of the technique in the diagnosis of latex allergy was first described by Ebo et al. (23). It was concluded that flow cytometric analysis of in vitro-activated basophils is not only highly sensitive and specific for the diagnosis of IgE-mediated allergy, but it was also demonstrated that the technique might be helpful to discriminate between clinical relevant and irrelevant latex IgE results resulting from sensitization to cross-reactive carbohydrate determinants (CCD). Cross-reactive carbohydrate determinants that have been demonstrated, on several occasions, to exhibit pronounced effects on the specificity of specific IgE quantification and to mimic allergy (71–73).
Since several independent groups have confirmed these data with sensitivities and specificities generally exceeding 90% (53, 74). In a study by Boumiza et al. (36) sensitivity for the CD63-based assay was only 50% when compared with 75% for the CD203c technique. However, this study has been commented and the conclusion of the authors that application of CD203c is a marked improvement over CD63 has, at least, to be considered as premature (75, 76).
Food. Flow-assisted diagnosis of IgE-mediated food allergy was first reported by Moneret-Vautrin et al. (77). From this study in patients with heterogeneous primary food allergies, the authors concluded that the flow cytometric assay gives comparable results to the leukotriene (LTC4) release test and was more efficient than the histamine release test. The technique also proved highly efficient in the diagnosis of IgE-mediated allergy from the fish parasite Anisakis simplex (60).
In individual patients, the technique confirmed diagnosis of primary food allergies such as anaphylaxis from sesame (Sesame indicum) seed and oil (78), the food additive guar gum (Cyamopsis tetragenoloba) (79), an oral allergy syndrome from papaya (Carica papaya) (80).
Finally, the technique has also been assessed in the diagnosis of secondary food allergies, i.e. food allergies that result from cross-reactivity to pollen allergens. According to history and healthy controls, the flow cytometric assay demonstrated a comparable sensitivity and specificity with skin tests and specific IgE for several birch pollen-associated food allergies such as carrot, celery and hazelnut (81). Almost similar results were obtained in a study on birch pollen-associated apple allergy (26). The latter paper, however, also demonstrated that validation of a diagnostic test is inappropriate when it failed to identify conditions that could have affected the outcome of the test. Actually, in a separate comparison between birch pollen-allergic patients with and without apple allergy, it was shown that cross-reactivity considerably reduced specificity of IgE quantification and (albeit to a lesser test) the flow cytometric technique.
Whether application of recombinant allergens will further improve the technique remains to be established. In a first experiment, with the Bet v 1 homologues Mal d 1, Api g 1 and Dau c 1 [the major allergens of birch (Betula verrucosa), apple (Malus domesticus), celery (Apium graveolens) and carrot (Daucus carota) respectively] sensitivity and specificity of the test varied between 65% and 75%, and 68 and 100% respectively (50). (For a comparison with natural extracts, see Table 2.)
Hymenoptera venom. Four studies have investigated flow cytometric analysis of in vitro-activated basophils in the diagnosis of anaphylaxis from wasp and bee venom. The first group to publish on the reliability of flow-assisted diagnosis of venom allergy reported an absolute sensitivity and specificity of the test with a good correlation between this method and mediator release tests (46). Almost similar observations on CD63 expression and histamine release were found by Lambert et al. (82). Unfortunately, robust interpretation from the data might to some extent be hampered by issues such as absence of clear dose-finding experiments and joint reporting of the results obtained for different hymenoptera venoms (vespula, polistes, hornets, bees, etc.).
In more recent series by Sturm et al. (58) and Erdmann et al. (49), the sensitivity and specificity of flow cytometric analysis of in vitro venom-activated basophils was comparable with or superior to more established diagnostic tests such as venom skin tests and specific IgE, with a sensitivity and specificity of 85–90%. Moreover, patients could be identified even after a long interval to the relevant sting reaction and despite a low IgE result.
From the Erdmann paper it appears that flow cytometric analysis of in vitro activated basophils failed to monitor successful venom immunotherapy and to predict the outcome of a controlled sting challenge (49). However, comparison between maximal and sub-maximal stimulation conditions demonstrated a significant decrease of basophilic CD63 expression during maintenance venom immunotherapy. Actually, after prolonged maintenance therapy of 3 years, for sub-maximal stimulation conditions, up to 60% of the patients had a negativation of their basophil activation test (D.G. Ebo, unpubl. obs.). However, ‘refractoriness’ in our whole-blood assay might not really represent a basophil property, but rather mirror less IgE that is cross-linked on the cell membrane because of decreased specific IgE and/or induction of blocking antibodies.
On several occasions, the technique has also been demonstrated to take the sting out of difficult cases with inconclusive IgE and skin test results (58, 61, 83) and to provide a tool for prediction of side effects from venom immunotherapy (61). A CD203c-based flow assay confirmed the diagnosis of hymenoptera venom allergy in 20 of 22 patients and demonstrated expression of this activation marker to be consistent with expression of CD63 (33).
Drugs and related compounds. Diagnosis of drug allergy is difficult, as a broad spectrum of different drugs (or metabolites) can elicit immune and non-immune-mediated pathologies with distinct and sometimes unclear pathomechanism. The causative structure (epitope) is frequently unknown, the result of an in vitro or in vivo test might not be predictive for the clinical outcome, and the reference test for diagnosis, the challenge test, is complicated and sometimes dangerous endeavour. Problems are frequently compounded by the fact that different drugs are administered simultaneously. Consequently, the availability of a safe, quick and reliable assay allowing simultaneous testing of different drugs would be more than welcome. Ideally, such a test should provide the physician with a tool that, apart from identification of the responsible compound(s), might also allow assessment of cross-reactivity and tailor safe alternatives.
Neuromuscular blocking agents. Evidence has accumulated that flow cytometry can contribute to the diagnosis of anaphylactic and anaphylactoid reactions from neuromuscular blocking agents that bear quaternary ammonium (NH) ions. In a series including 21 patients with definite anaphylaxis from these drugs, the sensitivity and specificity of a CD63-based basophil activation assay was 64% and 93%, respectively (51). More recently, comparable sensitivity and specificity figures were obtained in a study by Monneret et al. (24) and Sudheer et al. (27). In an own comparison between 14 patients suffering from anaphylaxis with profound hypotension and bronchospasm after administration of rocuronium and a positive skin test for rocuronium and eight individuals who tolerated administration of rocuronium and demonstrated a negative skin test for rocuronim, sensitivity and specificity of the test was 91.7% and 100% respectively. But two patients had to be excluded because of non-responsiveness to drug and positive control stimulation with anti-IgE. In line with the series of Refs (24, 27, 51), flow cytometry also proved helpful to identify cross-reactivity between rocuronium and other muscle relaxants (particularly with the other aminosteroid vecuronium) and to tailor a potentially safe alternative (D.G. Ebo, unpubl. obs.).
Beta-lactam antibiotics. In a comparative analysis between quantification of specific IgE and basophilic CD63 expression, in 58 patients suffering from skin-test proven β-lactam allergy and 30 healthy control individuals, sensitivity and specificity of the assays approximated 38% and 87% for IgE and 50% and 94% for the basophil activation test respectively (25). As demonstrated in Table 1 almost similar results were found in the study by Torres et al. (84).
Aspirin and non-steroidal anti-inflammatory drugs. Two studies from the same group have evaluated flow cytometric analysis of in vitro activated basophils in the diagnosis of ‘hypersensitivity’ from aspirin, metamizol and other non-steroidal anti-inflammatory drugs (NSAIDs) (85, 86). From the first study, sensitivity and specificity of the test for metamizol was 42% and 100% respectively. In the second study, the sensitivity for the different NSAID varied from 15% for metamizol and 55% for naproxen, whereas specificity generally exceeded 90%. Additional comprehensive studies are eagerly awaited to confirm these results. Particularly, because aspirin-induced respiratory as well as cutaneous reactions are typical pseudo-allergic manifestations that result from inhibition of cyclooxygenase-1 (COX-1) with subsequent depletion of prostaglandin E2 and unrestrained synthesis of cysteinyl leukotrienes and mediator release from mast cells and eosinophils (87, 88). Erdmann et al. (89) did not find the BAT to be reliable for the diagnosis of hypersensitivity from NSAID.
Miscellaneous. In isolated cases, the technique helped to identify the plasma expander hydroxyl ethyl starch (90), chemophor (the emulsifier of the intravenous cyclosporine formulation) (91), bovine serum albumin present in a semen culture medium for artificial insemination (92), omeprazole (93), different low-molecular-weight heparins (94, 95), the antiseptics povidone (96) and chlorhexidine (97), viscotoxins of mistletoe (Viscum album) (98), the dye patent blue (59) and the enzyme hyaluronidase (99) as the causative compound in patients with sometimes life-threatening anaphylactoid reactions. Moreover, in some of these patients the technique contributed to establish the individual therapeutic alternative and/or allowed identification of potentially cross-reactive structures.
Analysis of in vitro-activated donor basophils
Application of flow-assisted analysis and quantification of in vitro-activated basophils can also be applied on cells drawn from non-allergic and/or allergic donors. In a first application, generally referred as a passive sensitization procedure, basophils (stripped) of non-allergic donors, prior to allergen challenge, are incubated with serum of the patient (77, 100). Passive sensitization protocols to detect allergen-specific IgE in the patients’ serum, however, are laborious and difficult to standardize and to reproduce as it is highly dependent on the basophil releasability of the individual donor.
A second application comprises detection of mediator-releasing antibodies in a typical form of chronic urticaria, i.e. auto-immune urticaria. The auto-immune subclass of chronic idiopathic urticaria has been characterized by the presence of cell-activating IgG autoantibodies against the IgE molecule or α-chain of the high-affinity FcɛRI on basophils and mast cells. Currently, these antibodies are usually detected by autologous serum skin tests and confirmed by histamine release studies [for review, see Ref. (101)]. As already addressed above, a particular application of the flow cytometer is the autoimmune basophil activation test. In these settings, basophils of allergic or non-allergic individuals are stimulated with patients’ serum in order to detect the presence of basophil-activating autoantibodies. At present, this application has been reported in three studies. First, the paper by Wedi et al. (102) who, with the use of an atopic donor, demonstrated increased CD63 expression in 70%, 45% and 35% of the patients with a positive and negative autologous serum test and controls respectively. Secondly, the report by Gyimesi et al. (103) who demonstrated a strong correlation between the ability of patients’ sera to raise a positive autologous serum skin test and CD63 expression by the basophils of atopic donors. Recently, these data were confirmed in a third study by De Swerdt et al. (104), who found positive basophil activation in 68% of chronic idiopathic urticaria patients with a positive autologous serum skin test and 41% of the chronic idiopathic urticaria patients with a negative autologous serum skin test.