Comparison of two basophil activation markers CD63 and CD203c in the diagnosis of amoxicillin allergy.

BACKGROUND: To confirm allergy to beta-lactam (BL), a basophil activation test in flow cytometry based on CD63 up-regulation was described. CD203c is a more recent basophil activation marker and up to day there is no consensus about which marker is the more sensitive one. CD203c has not yet been evaluated in the diagnosis of BL allergy. OBJECTIVE: The aim of the study was to compare the reliability of CD203c to CD63 for the diagnosis of amoxicillin (AX) allergy, which is nowadays the most frequent BL allergy. METHODS: Twenty-seven patients with an immediate positive skin test (ST) to AX, 20 had had anaphylaxis with AX and 7 had urticaria and/or angioedema, were compared with 14 controls with no allergy to BL and to six patients with delayed positive ST to AX. RESULTS: In the anaphylaxis group, AX induced up-regulation of CD203c in the basophils of 12 patients out of 20 (60%) and of CD63 in four patients (20%) (P<0.02). Two patients out of seven with urticaria or angioedema had a positive result with CD203c and CD63. In patients who had anaphylaxis, ampicillin (AMP) induced CD203c up-regulation in eight out of 12 (67%) patients tested, and CD63 up-regulation in 4 out of 12 (33%) (all patients who had anaphylaxis could not be tested with AMP). False-positive results were observed with CD203c as well as CD63, and for 10 patients indeed this was confirmed by a negative drug provocation test. The origin of conflicting results between CD63 and CD203c might be at least the targeting of basophils based on anti-IgE labelling. Among IgE(+) gated cells, by means of CD33, a marker of monocytes, a contamination up to 50% by monocytes was detected. In contrast to CD63, CD203c is an activation marker specific of basophils with a basal low-level expression in resting basophils. Thus, IgE and CD203c double targeting of basophils avoids the contamination by monocytes. CONCLUSION: CD203c seems to be a more sensitive activation marker of basophils than CD63 for the diagnosis of amoxicillin allergy.

Autoantibodies to phospholipids and to the coagulation proteins in AIDS.

In HIV-1 infection, an increased prevalence of anticardiolipin autoantibodies (aCL) and lupus anticoagulant (LA) has been described. In order to see if these antibodies are isolated or, like in autoimmune diseases, associated with hematological disorders and with antibodies to other phospholipids and to proteins of coagulation, we investigated 3 groups of patients: 1. 342 HIV-1 infected patients, 2. 145 control patients including 61 systemic lupus erythematosus (SLE) patients, 58 patients with a connective tissue disease, 15 patients with stroke, 11 patients with syphilis and 3. 100 blood donors. In HIV-1 infection antiprothrombin (aPrT) antibodies were present in 2% of patients, the prevalence of antiphosphatidylcholine antibodies (aPC) (50%) was almost as high as aCL (64%), and 39% had both antibodies. Absorption on liposomes of the latter revealed an heterogeneous mixture of aCL and aPC or cross-reacting antibodies. In contrast with SLE, anti-beta 2-glycoprotein I (4%), LA (1%), biological false positive test for syphilis (0.3%), thrombosis (p < 0.001) were uncommon. In HIV-1 infection, antiphospholipid antibodies do not associated with features linked to them in SLE or syphilis.

[Conclusions of the first French workshop of standardization of anticardiolipin antibody determination associated with autoimmune pathology]. / Conclusions du 1er Atelier francais de standardisation du dosage des anticorps anticardiolipine associés à la pathologie autoimmune.

Anticardiolipine antibodies are a marker of the antiphospholipid syndrome. They are detected by Elisa. Despite its simplicity, the results obtained by this assay are not always reproducible despite several worldwide standardizations and the distribution of calibration sera by EN Harris. On 4 December 1992, the First French Anticardiolipin Antibodies Standardization Workshop was held in Paris. Eight coded standards were sent to 33 laboratories in France, one in Switzerland and one in Luxembourg. Some of them used several assays. Agreement between qualitative results was good for samples with high and moderate level of antibodies. But the results expressed in IgG antiphospholipid units were scattered, the interlaboratory coefficient of variation for each of the eight standards was higher than 70%, though the within-run coefficient of variation had a median value of 10%. Assays using bovine serum for the blocking buffer and for the dilution buffer seemed to give more reproducible results than assays using purified bovine serum albumin (p < 0.001). For only nine assays out of 38 which expressed results quantitatively, was a linear regression observed between the dilutions of a sample and the results expressed in IgG antiphospholipid units. Absence of linearity is an obstacle which must be overcome if we wish to compare quantitative results obtained by different laboratories.

Association of antimitochondrial antibodies type 5 and anti-beta 2 glycoprotein I antibodies in the antiphospholipid syndrome.

OBJECTIVE: To evaluate if antimitochondrial type 5 antibodies (AMA5) might be included among antiphospholipid syndrome (APS) markers. METHODS: In a retrospective study, blood variables of 48 patients with AMA5 were analyzed in relationship with clinical and biological markers of APS and systemic lupus erythematosus (SLE). RESULTS: We observed a high prevalence of false biological test for syphilis (95%), lupus anticoagulant (LAC) (71%), anticardiolipin antibodies (aCL) of IgG (71%) and IgM (75%) isotype, positive direct Coombs' test (54%), thrombocytopenia (52%), anti-B2 glycoprotein I antibodies (38%). Twenty-nine patients (61%) had at least one clinical manifestation of APS; 42% had recurrent arterial and/or venous deep thrombosis and 21% had recurrent fetal loss. But, for 2 patients, AMA5 were the sole detected immunological marker. Moreover, SLE was observed in 35% of the patients. These were different from 100 control patients with SLE with the respect to skin involvement and dsDNA antibodies which were less frequent (p < 0.01) and aCL, LAC, false biological test for syphilis (p < 0.001), positive direct Coombs' test and thrombocytopenia (p < 0.05) which were more frequent. CONCLUSION: Our data suggests (1) AMA5 is another marker of the APS (2) in patients with SLE, AMA5 seems to be a marker of a subset of SLE. This appears to justify the routine detection of these antibodies.

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

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 B asophils' A ltered S urface I mmunofluorescence is detected by flow C ytometry (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.