Validation of a flow cytometric assay detecting in vitro basophil activation for the diagnosis of muscle relaxant allergy

Validation of a flow cytometric assay detecting in vitro basophil activation for the diagnosis of muscle relaxant allergy Nisen Abuaf, MD, PhD, Bakoliarisoa Rajoely, MD, Ezzedine Ghazouani, PhD, David A. Levy, MD, Catherine Pecquet, MD, Habib Chabane, MD, and Francisque Leynadier, MD Paris, France Background: Anaphylactic reactions during anesthesia are mainly the result of muscle-relaxant (MR) drugs. Skin tests, serologic detection of specific IgE, and in vitro leukocyte histamine release are used to investigate MR allergy. Objective: We describe a new assay that is based on the detection by flow cytometry of the altered expression of plasma membrane molecules of MR-activated basophils. Methods: For this assay, which we have named the BASIC assay, basophils are incubated in vitro with MR, after which they are fixed and then triple labeled with fluorescein-conjugated anti-CD63, tandem dye R-phycoerythrin-cyanin 5.1 conjugated anti-CD45, and R-phycoerythrin conjugated anti-IgE. The resulting Basophils’ Altered Surface Immunofluorescence is detected by flow Cytometry (BASIC). Results: Forty-one patients who had an allergic reaction during general anesthesia and 23 control subjects without such a history were studied. All included subjects’ basophils were tested in the BASIC assay with at least 4 MR: suxamethonium, gallamine, vecuronium, and pancuronium. After reaction of the basophils of the MR-allergic patients with MRs, increased surface expression of CD63 and CD45 and decreased expression of IgE were detected. Increased expression of CD63 was observed most frequently and it was stronger than the alteration of the 2 other markers. Cross-reactivity between MRs commonly occurred. MRs diluted 10–1 activate the basophils of the control subjects, suggesting that at relatively high concentrations MRs are also nonspecific basophil activators. Conclusion: In the diagnosis of MR allergy, the BASIC assay has a good specificity but a low sensitivity, and it correlates strongly with skin test results. It is currently appraised for the diagnosis of anaphylactic reaction induced by other classes of drugs. (J Allergy Clin Immunol 1999;104:411-8.) Key words: Myorelaxant drugs, anaphylactic reactions, drug allergy, basophils, surface molecules, flow cytometry

Anaphylactic reactions (AR) during anesthesia are mainly the result of muscle-relaxant drugs (MRs).1-5 As part of the preoperative investigation of patients with a

From the Service d’Immunologie et d’Hématologie Biologique and the Service de Médicine Interne et d’Allergologie, Hôpital Rothschild Assistance Publique, Hôpitaux de Paris, Centre Hospital-Universitaire Saint-Antoine, Université Pierre et Marie Curie, Paris, France. Received for publication Mar 1, 1999; revised Apr 26, 1999; accepted for publication May 6, 1999. Reprint requests: Nisen Abuaf, MD, PhD, S. Immunologie & Hématologie, Hôpital Rothschild, 33, Boulevard de Picpus, 75571 Paris Cedex 12, France. Copyright © 1999 by Mosby, Inc. 0091-6749/99 $8.00 + 0 1/1/99859

Abbreviations used AR: Anaphylactic reaction BASIC: Basophils’ altered surface immunofluorescence detected by flow cytometry FcεR1: High-affinity receptor for IgE LHR: Leukocyte histamine release assay MR: Muscle-relaxant drug s: On the surface of the plasma membrane

history of a previous intraoperative AR, the detection of MR-specific IgE antibodies is essential. The “gold standard” for this is the immediate skin test response to a suspected MR. However, the predictive value of skin tests with MRs is relatively low.1 For example, 10% of women tested before receiving general anesthesia have been reported to have either a positive or doubtful skin test to one or more MRs.1 This result is in contrast to the fact that intraoperative ARs occur in women with a frequency of only about 0.02%. Solid-phase RIA with MR or quaternary ammonium–bearing molecules coupled to Sepharose can be used as an alternative to skin testing to detect MR-specific IgE antibodies in serum.2-4 However, currently available RIAs have lower predictive values than skin tests do. For example, in one prospective study a high rate of falsepositive and false-negative results was observed with a radioallergosorbent test to suxamethonium, alcuronium, and thiopental.5 Thus skin tests are still considered to have better predictive value than RIAs6; nevertheless, they are not used routinely by anaesthetists. A laboratory-based assay that had greater specificity and predictive value than skin testing would be helpful. An alternative to RIA would be a test to detect IgE-sensitized polymorphonuclear basophils in vitro. When IgE-sensitized human basophils are activated by specific allergen, the altered expression of both CD45 and IgE on their plasma membrane can be detected by flow cytometry.7 We previously reported that this assay could be used to detect allergic sensitization to inhalant allergens.8 We had also reported that this assay was, however, not sufficiently sensitive for the diagnosis of IgEmediated drug-induced allergies.9 Recently we have improved the assay in 2 ways. First, we added a third basophil surface marker, namely, anti-CD63 antibodies, which significantly increased the sensitivity of the assay. 411

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TABLE I. Clinical data on 33 MR-allergic patients and 29 control patients Groups

Allergic to Vecuronium Suxamethonium Rocuronium Atracurium Pancuronium Alcuronium Control patients Group 3 Preanesthesia Healthy

No.

Sex ratio (male/female)

Age (y)

(Range)

Clinical history (group 1/group 2)*

15 8 6 2 1 1

1:14 2:6 2:4 1:1 1:0 0:1

46 45 45 42 58 38

(34-75) (32-61) (24-60) (40-44)

11/4 7/1 6/0 2/0 1/0 1/0

8 14 7

2:6 6:8 5:2

47 44 36

(16-68) (25-73) (26-49)

— — —

*Ratio of patients in group 1 (typical anaphylactic reaction) to group 2 (atypical reaction) (see Material and methods).

Second, to avoid activation of the basophils because of the cross-linking of surface molecules when antimarker antibodies are added, we now fix the cells just after their incubation with allergen, before we add the antibodies. We named this modified assay the Basophil Altered Surface Immunofluorescence detected by flow Cytometry assay (BASIC). The aim of the current study was to validate the BASIC assay for the diagnosis of allergy to MRs, in comparison with skin testing.

MATERIAL AND METHODS Patients Forty-one patients referred by the anesthetists to the Centre d’Allergie of the Hôpital Rothschild between 1995 and 1997 for evaluation of a history of an AR during general anesthesia were included. These subjects were divided in 3 groups: group 1, 28 patients who had had a typical AR characterized by tachycardia, hypotension, urticaria, or angioedema less than 5 minutes after the injection of an MR; group 2, 5 patients whose intraoperative reaction was not typical of an MR-induced AR or was delayed in onset (beginning more than 15 minutes after injection of the MR); and group 3, 8 patients, considered as control subjects, whose allergic reaction was not related to the injection of an MR. Five patients were allergic to latex. Included as additional control subjects were 14 patients having negative skin test results to MRs in the preoperative (preanesthesia) evaluation and 7 healthy adult volunteers not skin tested. Both groups had no history of a previous intraoperative allergic reaction. The study protocol was in accordance with the local ethical committee guidelines, and all subjects gave their consent before being included. Table I lists the subjects’ sexes and ages and for groups 1 and 2 the MR responsible for the reaction as well as its severity. Vecuronium, the MR most frequently used in France, was the agent that had most frequently induced an AR in the patients. The patients’ mean age was 45.4 years and 79% were female.

Skin tests Intradermal skin tests were performed at least 6 weeks after AR as previously described.10,11 Through a 25-gauge hypodermic needle, 0.05 mL of commercially available MR diluted 1:1000 in sterile saline solution containing 0.5% phenol (Stallergènes, Fresnes, France), was injected on the volar surface of the forearms. If skin test result was negative at this dilution, a second test was done with 1:100 diluted MR. Prick tests with codeine phosphate solution (9%)

and phenol-saline solution were used as positive and negative controls, respectively. The reactions were read 20 minutes later by measuring the diameter of the wheals and flares. A test result was considered to be positive when the diameter of the resulting wheal and accompanying flare were at least 6 and 10 mm, respectively, and the positive and negative controls were within the usual limits.

MRs Commercially available pancuronium (Pavulon 2 mg/mL = 2.73 mmol/L), vecuronium (Norcuran 4 mg/mL = 6.27 mmol/L), and rocuronium (Esmeron 10 mg/mL = 16.4 mmol/L) were purchased from Organon Teknika, Fresnes, France; suxamethonium (Celocurine 10 mg/mL = 20.9 mmol/L) was obtained from Pharmacia, SaintQuentin en Yvelines, France; and gallamine (Flaxedil 20 mg/mL = 22.4 mmol/L) was obtained from Spécia, Paris, France. For skin tests, MRs were diluted to 1:10 (10–1), 1:100 (10–2), and 1:1000 (10–3), in sterile phenol-saline solution immediately before being used. For in vitro assays MRs were diluted in the dilution buffer, 2 mmol/L magnesium chloride and 1.2 mmol/L calcium chloride in PBS. Bovine serum albumin 2 g/L (Eurobio, Les Ulis, France, ref 0126) was added to this buffer immediately before it was used.

Antibodies FITC-conjugated anti-CD63 monoclonal antibody and tandem dye R-phycoerythrin-cyanin 5.1 conjugated anti-CD45 mAbs were purchased from Coulter-Immunotech, Coultronics, Margency, France. Biotinylated goat antihuman IgE polyclonal antibody and R-phycoerythrin conjugated streptavidin were purchased from Vector Laboratories, Burlingame, Calif.

BASIC assay Basophil-enriched leukocytes were isolated from lithium heparin–anticoagulated peripheral blood (Vacutainer 7-mL glass tubes, Becton Dickinson, Le Pont de Claix, France) by centrifugation at 500g for 20 minutes at 20°C on a layer of Ficoll (d = 1.077) (Eurobio, Les Ulis, France). One hundred microliter aliquots of the leukocyte suspension containing 106 leukocytes were mixed with 100 µL of antigen or dilution buffer and then incubated 15 minutes at 37°C, after which the cell suspensions were fixed by the addition of 50 µL of 4% paraformaldehyde in PBS.

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A

B

C

D

FIG 1. Detection by flow cytometry of activated basophils (BASIC assay). Labeling by anti-IgE of leukocytes isolated by centrifugation on layer of Ficoll distinguishes at least 2 populations, the larger but poorly labeled contains lymphocytes and monocytes, and the smaller (arrow), strongly labeled (IgEbC) contains basophils (A). Gating of strongly labeled IgE shows that most of cells stuck on axis (arrow) do not express surface CD63 (C). After activation strongly labeled IgE decreased (B) and surface expression of CD63 and CD45 increased (D). PE, Phycoerythrin.

The basophils were then triple labeled by adding 10 µL of FITCconjugated anti-CD63 antibody, 10 µL of tandem dye R-phycoerythrin-cyanin 5.1 conjugated anti-CD45 antibody, and 50 µL of biotinylated anti-IgE antibody, diluted 1:50. After being mixed and incubated for 30 minutes at 4°C, the cell suspensions were washed and pelleted by centrifugation at 500g for 10 minutes at 20°C. After this, 50 µL of R-phycoerythrin conjugated streptavidin diluted 1:100 was added and the cells were incubated for 20 minutes at 4°C. Then the cells were washed again. Flow cytometric analysis of surface markers of basophils was performed at 488 nm on an Epics XL flow cytometer equipped with a 15-mW argon ion laser lamp (Coulter, Coultronics, Margency, France). On the histogram defined by forward scatter and side scatter the initial gating of cells was done by a bit map around lymphocytes and monocytes. The second gating was done, shown in the histogram where IgE-labeled cells were represented (Fig 1), around the brightest cells (IgEbC). These cells had previously been identified as basophils.7 In each assay the mean fluorescence intensity was measured on at least 200 basophils. For CD45 and CD63, the results are expressed as the percent change in the mean fluorescence intensity of basophils, according to

the following formula: ∆Fluorescence (%) = 100 × (A – B)/B, where A is the mean fluorescence intensity (channel) observed with basophils that were incubated with allergen and B (blanc value) is the mean fluorescence intensity (channel) of basophils that were incubated in dilution buffer without allergen. For strongly labeled IgE (IgEbC), the results are expressed in percent change of the bright cells labeled by anti-IgE according to the following formula: ∆IgEbC (%) = 100 × (A – B)/B, where A is the percent of bright cells observed after incubation with the allergen and B (blanc value) is the percent of bright cells observed after incubation with the dilution buffer. Relative to the buffer control, an increase of 15% of the surface expression of CD63 or CD45 or a decrease of 15% of strongly labeled IgE was observed in fewer than 10% of control subjects’ basophils (90th percentile) incubated with MRs diluted 1:100. Therefore an alteration of 15% of a marker was considered to be significant criteria for basophil activation.

Statistical analysis The Spearman rank test was used for nonparametric correlation analysis; chi-square and a 2-tailed Fisher test were used for com-

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FIG 2. Dose-response curves resulting from challenge of patients’ basophils with MR to which they were allergic. Highest concentration for each drug is that obtained at 1:10 dilution of commercially available solution. For sCD63 and sCD45 values are in percentage of increase of mean fluorescence, and for bright cells labeled by anti-IgE (IgEbC), values are in percentage of decrease in number of cells. Control (C) was incubated with buffer.

parison of qualitative results. The clinical sensitivity of the assay was calculated as the ratio of the number of MR-allergic patients with a positive test result to the total number of MR-allergic patients tested. The clinical specificity was calculated as the ratio of the number control subjects with negative test results to the total number of control subjects tested. The odds ratio for a test was calculated as the ratio of the number of allergic patients with a positive test result relative to the number of allergic patients with negative test results divided by the ratio of the number of control subjects with a positive test result relative to the number of control subjects with a negative test result.

RESULTS Flow cytometric analysis of triple-labeled basophilenriched leukocyte suspensions revealed at least 2 distinct cell populations. The smaller population consisted of cells that were brightly labeled by fluorescent anti-IgE antibodies; we refer to these cells as “IgEbC” (Fig 1, A). They were previously identified as basophils.7 The larger population (also seen in Fig 1, A) consisted of dimmer fluorescent cells that were poorly labeled by anti-IgE and includes lymphocytes, monocytes, and a small number of eosinophils. Both populations were similarly labeled by anti-CD45 antibodies (Fig 1, A). Gating of brightly labeled IgE and analysis of these cells showed that most of these cells did not express surface (s) CD63 (Fig 1, C). In allergic patients in vitro basophil activation induced by the MRs revealed a dimmer fluorescence after antiIgE labeling and a decrease in the number of brightly labeled IgE (Fig 1, B compared with A). In contrast, the expression of other surface molecules, sCD45 and

sCD63, especially the latter, was significantly increased (Fig 1, D compared with C). After activation, a fraction of the basophils was shifted from the brightly labeled IgE population to the larger and dimmer fluorescent population. This fraction of basophils could not be distinguished from other leukocytes and therefore could not be gated, making it impossible to measure the increase of sCD45 and sCD63 on them. The optimal dilution of the MR giving the strongest response in the BASIC assay varied widely from patient to patient and from MR to MR. Fig 2 shows the MR dose-response curve obtained in the BASIC assay with leukocytes from typical patients, each allergic to a different MR. Nevertheless, the 10–2 dilution was the one most often inducing an in vitro activation of allergic patients’ basophils. Only MRs diluted 10–2 were used when basophils were obtained in a limited number, and to standardize all the comparisons were made with results obtained at this dilution. Even if in few allergic patients a positive BASIC assay was obtained only when basophils were incubated with 10–1 diluted MR, the lack of specificity of results obtained do not allow us to use this dilution in the diagnosis of AR to an MR. Thus in 7 normal controls tested with 4 MRs (28 assays), vecuronium, pancuronium, gallamine, and suxamethonium at the 2 dilutions 10–2 and 10–1, a positive BASIC test (>15% ) was observed in 5 assays versus 11 assays for sCD45, 2 assays versus 14 assays for sCD63, and 1 assay versus 1 assay for brightly labeled IgE. Six of 7 control subjects’ basophils reacted with 10–1 diluted vecuronium, 4 react-

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TABLE II. BASIC assay and skin test results in MR-allergic patients and controls BASIC assay (positive tests/total)* Groups

Allergic Group 1 Group 2 Control Group 3 Preanesthetic Normal

Skin test (positive/total) (MR dilution)

IgEbC

sCD63

sCD45

10–2

10–3

21† 5

11/21 (52%) 1/5 (20%)

13/21 (64%) 2/5 (40%)

9/21 (43%) 2/5 (40%)

21/21 (100%) 4/5 (80%)

12/21 (57%) 2/5 (40%)

8 14 7

2/32 (7%) 3/56 (5%) 2/28 (7%)

6/32 (19%) 4/56 (7%) 0/28 (0%)

1/32 (4%) 7/56 (12%) 3/28 (11%)

12/32 (38%) 0‡ ND

2/32 (6%) 0 ND

No. of patients

IgEbC, Brightly labeled IgE; ND, not done. *Alteration of marker >15% is recorded positive result in BASIC assay. †Data for groups 1 and 2 are those observed with MR that induced anaphylactic reaction during anesthesia. Patients not tested with corresponding MR are excluded. For other groups results are those observed with each of 4 MRs. For these groups number of results exceeds by 4 times the number of patients. ‡In preanesthesic evaluation group, potential subjects with positive MR skin test result were excluded from study.

ed with pancuronium, 2 with suxamethonium, and 1 with gallamine. All the patients who had had a typical allergic reaction to MR (group 1) had a positive response to at least 2 of the 3 cell surface markers. sCD63 was the marker most frequently altered, followed closely by the other 2 markers (Table II). Concurrent alteration of the expression of all 3 was rather unusual, especially when the degree of activation was only moderate. Of the 10 patients whose cells showed the strongest alteration of the surface markers, simultaneous alteration of all 3 markers occurred in only 5 cases. In the 27 patients and controls with only a moderate or a weak response in any marker, simultaneous alteration of all 3 markers was observed in only a single case. In control subjects no more than 1 of the 3 markers was ever altered significantly on challenge of their leukocytes with the 4 MRs. In the 116 assays of leukocytes from 29 control subjects including normal subjects, preoperative patients, and allergic patients in group 3, and each subject tested with 4 MRs, simultaneous alteration of at least 2 markers was not observed. Likewise, when potentially allergic patients’ basophils had a significant change in the expression of only 1 the 3 markers, they were found not to be clinically allergic to the MR used in the test. All patients in group 1 and 80% of those in group 2 had a positive intradermal skin test result to a 1:100 dilution of the MR that had induced their reaction, whereas only 3 of 8 of the non-MR allergic group 3 patients had a positive skin test result to any of the 4 MRs (Table II). With the MR diluted 1:1000, the number of positive test decreased but the clinical specificity increased. Because preoperative patients with a positive MR skin test result were excluded from the study, none of those included had a positive skin test response to any of the 4 MRs. The normal control subjects were not skin tested. There were strong correlations (P < .001) between skin test responses expressed either as diameter of the resulting wheal or as the highest dilution of the MR still giving a positive test result and alterations in each of the 3 cell surface markers (Fig 3, A). In the Spearman rank

correlation test, the alteration of sCD63 (r = 0.53) or brightly labeled IgE (r = 0.47) correlated better than the alteration of sCD45 (r = 0.39). When skin test results were compared with the concurrent alteration of 2 markers, the couple sCD63 and brightly labeled IgE correlate the best (Fig 3, B). Patients allergic to one MR frequently react in skin tests to closely related MRs.10,11 In the current study there were strong correlations between skin test results with suxamethonium and gallamine and with vecuronium and pancuronium (Table III). There were also strong correlations in the Spearman rank test (P < .001) between the level of alteration of markers to these same pairs of MRs in the BASIC assay (Table III). In the same way positive reactions to pairs of MRs were found in 46% to 75% of assays, depending of the pairs of MRs (results not shown). In most cases the MR giving the strongest reaction to those tested was the one to which the patient was known to be allergic. However, the basophils of 2 of 10 patients in group 1 allergic to vecuronium reacted more strongly to pancuronium than to vecuronium, and the basophils of 2 other patients reacted more strongly to both pancuronium and suxamethonium. Results obtained in the BASIC assay were analyzed according to the history of patients (Table II). The clinical history of the patient had not been known by the persons who worked on the BASIC assay and the study was done in blind. The basophils of the 33 patients with typical or atypical AR (groups 1 and 2) were reacted with at least 4 MRs: vecuronium, pancuronium, gallamine, and suxamethonium. The cells of 2 of the 6 rocuronium-allergic patients were also reacted with that MR, whereas the cells of 3 other patients, allergic to either atracurium or alcuronium, were not reacted with the corresponding MR. Of 28 patients of group 1, basophils of 21 patients were tested with the corresponding MR. The strongest alteration for the 3 surface markers of the basophils were observed in patients with a typical history of an AR (group 1) (Table II) and they were usually induced by the MR that had induced the AR. In more

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A

B FIG 3. Alteration of surface markers induced by MRs in relation to result of skin test. Positive skin test was observed in 35 cases with the MR diluted 1:1000, in 46 cases with the MR diluted 1:100, and in 7 cases with the MR diluted 1:10; in 95 cases skin test result was negative. Number of results exceed number of patients because each patient was tested with at least 4 MRs. Result of BASIC assay and of skin test are compared for same MR. Percentage of positive assays are indicated on the top of columns. For all markers, BASIC assay was recorded as positive when alteration was higher than 15% (A). B, BASIC assay was recorded as positive when at least 2 markers were concurrently altered. IgEbC, Brightly labeled IgE.

than half the patients the MR inducing the AR gave a positive result in the BASIC assay. The basophils of the 5 patients in group 2 (ie, those with atypical reactions) were less likely to respond significantly to the responsible MR. The sensitivity of the BASIC assay was low, but specificity was high (Table IV), and false-positive results in nonallergic subjects were scarce. When at least 2 surface markers were altered concurrently, this assay displayed equal sensitivity and specificity to that of the skin test

performed at 10–3 diluted drug. Skin test at 10–2 diluted MR had a higher sensitivity but a lower specificity.

DISCUSSION When reacted in vitro with the muscle relaxant that was responsible for their ARs, basophils from MR-allergic patients altered the expression of the 3 membrane molecules, CD63, CD45, and IgE, that we chose as probes for cell activation.

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TABLE III. Cross-reactions between MRs in BASIC assay and in skin tests BASIC assay (Spearman rank correlation)* Cross-reaction

Suxamethonium-gallamine Vecuronium-pancuronium Suxamethonium-pancuronium Suxamethonium-vecuronium Gallamine-pancuronium Gallamine-vecuronium

IgEbC

sCD63

0.71† 0.61† 0.35† 0.44† 0.41† 0.52†

0.61† 0.78† 0.21‡ 0.34† 0.59† 0.52†

Skin test*

0.72† 0.74† 0.22‡ 0.11 –0.04 –0.01

IgEbC, Brightly labeled IgE. *Correlation coefficients are similar using diameter for wheal or logarithm of highest dilution of MR giving positive skin test result. †P < .001. ‡P < .05.

CD63 is a tetraspan granule protein first found in platelet lysosomal granules, and its expression on the plasma membrane was found to be increased after platelet activation.12 In resting basophils CD63 is located on the intracytoplasmic granule membrane.13 After activation of basophils, the granule membrane fuses with the cell’s plasma membrane. The CD63 is translocated as a result, and its expression on the plasma membrane is increased. The kinetics of the increased CD63 expression and the kinetics of the accompanying histamine release are identical, and there is a strong positive correlation between these 2 events. Because platelets might adhere to the activated basophils, a fraction of sCD63 might come from platelets. However, allergic patients’ platelets, incubated in vitro with aeroallergens or drugs, did not express sCD63 (unpublished results). Because in these experiments sCD63 was measured on the free platelets, sCD63 expression by the subpopulation of platelets bound to the basophils cannot be excluded. CD45, the leukocyte common antigen, is a receptorlinked protein tyrosine phosphatase. On T lymphocytes the extracellular domains of CD45 is expressed in several different isoforms, the result of alternative messenger RNA splicing of exons 4´, 5´, and 6´, which code for this domain.14 CD45RA is found on virgin T cells and CD45RO on memory T cells. CD45RO associates with TCR-CD3 and CD4/8, whereas CD45 RA does not. Plasma membrane CD45RO but not CD45RA can be detected on basophils and neutrophils. Allergen-induced upregulation of the expression of CD45RO closely parallels that of CD45.8 There is currently no good explanation for the decrease in the number of IgE-positive basophils after antigen activation. Bridging and internalization of FcεR1 or up-regulation of activation molecules that could associate with this receptor might decrease by steric hindrance the binding of anti-IgE antibody to IgE on the cell surface. Alteration of the expression of cell surface molecules is not limited to the three that we have studied. It appears

TABLE IV. Sensitivity and specificity of cell surface markers and skin test for diagnosis of clinically significant MR allergy Evaluation of test* Test

Sensitivity

BASIC Brightly labeled IgE sCD63 sCD45 Brightly labeled IgE + sCD63 Brightly labeled IgE + sCD45 sCD63 + SCD45 2 of 3 markers Skin test (MR 1:100) Skin test (MR 1:1000)

0.52 0.64 0.43 0.36 0.30 0.23 0.55 1 0.57

Specificity

0.96 0.93 0.93 1 1 1 1 0.63 1

Odds ratio

26 24 10 >† > > > > >

*Sensitivity was calculated on results observed in 21 patients from group 1 (typical anaphylactic reaction). Specificity was calculated on results observed in control groups (Table II). For skin test, specificity was calculated on 8 patients of group 3. †Odds ratio for these results are too high (infinity) to be estimated because 100% of subjects had either positive or negative results.

to be a general phenomenon. For example, it has been reported for the adhesins CD11b, CD11c, and CD62L (Leu 8).15 Up-regulation of adhesins on the surface of basophils leads these cells to adhere to vascular endothelium.16,17 As a consequence, the BASIC assay cannot be used for the detection of activation of basophils in vivo because they are lost from the circulation within seconds of the intravenous infusion of allergens. The poor correlation in the alteration of the three basophil surface molecules that we probed after activation by antigen and the large differences between subjects suggest that alteration of the surface expression of these molecules is not linked. Only when activation is strong are all three markers altered significantly. Kinetic analysis has revealed that up-regulation of CD11b on basophils proceeds more rapidly than histamine release, whereas down-regulation of CD62L is much slower, reaching a plateau only at 120 minutes after stimulation.15 These facts suggest that improvement in the BASIC assay should not be limited to only one surface marker nor only to the three that we have used. The results obtained with the BASIC assay for MR allergy in the current study are similar to those obtained with the in vitro leukocyte histamine release assay (LHR).18 In both assays the optimal dilution of the MR was 1:100, and the sensitivity in the LHR assay (65%) was about the same as with the BASIC assay. All the patients with a positive LHR or BASIC assay also had a positive skin test to MR, although the skin test appears to be more sensitive than either of these assays. In contrast to the BASIC assay, with the LHR assay the cells of control subjects do not react to MR at a dilution of 1:10. Although some similarities with the published results exist, we cannot determine yet whether in the investigation of drug-induced allergy the BASIC assay is better

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than LHR. The sensitivity and the specificity of these tests need to be compared to each other in a blind assay. The heterogeneity of the determinants of allergenic drugs is well known and this seems to be true for MRs also.19 The cross-reactions observed by the BASIC assay and by skin tests seems to confirm the heterogeneity of the resulting IgE antibodies.10,11 Patients who react to 4 MRs are likely to have antibodies that react with the quaternary ammonium group, whereas patients who react to only 1 or 2 MRs are likely to have antibodies specific for other structures on these agents. Therefore in vitro assays based on the detection of antibodies specific for the ammonium group might not detect allergies where the antibodies only have other specificities. Cross-linking of FcεR1-bound IgE by a MR activates the basophil. The existence of this mechanism in MRallergic patients was confirmed by the discovery of MRspecific IgE antibodies.2 This does not, however, exclude the possibility of a second, non-antibody-mediated pathway of basophil activation by MRs. It was thought that the results with an undiluted or slightly diluted MR in skin tests could be the result of a nonspecific reaction.11 Currently there is no consensus on which dilution of MR to use for the result to be considered immunologically specific. In the current study even a 1:100 dilution of the stock MR sometimes gave a false-positive result to a skin test, whereas a 1:1000 dilution did not. In the BASIC assay a 1:10 dilution of the MR appeared to yield a nonspecific, nonimmunologic, secretagogue-like result. With use of the chemotactic oligopeptide formylmethionyl-leucylphenyl-alanine, it was shown that the kinetics of basophil histamine release and alteration of the expression of cell surface molecules were identical.13,15 Looking for new MRs, we screened synthetic tetrapeptides; some of them were very potent secretagogues and they activated basophils of patients and controls, but they were efficient only at concentrations higher than 1 mmol/L (Rivaille, unpublished results). These results indicate that nonimmunologic histamine-releasing agents can also induce alteration of the expression of cell surface molecules. Therefore our BASIC assay does not allow the determination of whether an anaphylactic or anaphylactoid reaction was caused by an allergen or by a nonspecific histamine-releasing agent. This is not, however, a handicap in the investigation of such druginduced reactions because some drugs act only as nonspecific histamine releasers. This contrasts with tests for specific IgE antibodies that are not efficient for the investigation of reactions induced by non-IgE-dependent histamine-releasing drugs.

J ALLERGY CLIN IMMUNOL AUGUST 1999

Although less sensitive than the skin test, the BASIC assay will still be useful in the investigation of perioperative anaphylactic or anaphylactoid reactions because it is more specific than skin testing. We are currently studying the efficiency of the BASIC assay for the diagnosis of allergies to other drugs.

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