The adverse "allergic" drug reaction can be distinguished in immediate(IR)and delayed type(DR),based on the time between the drug administration and the onset of the reaction.IR occurs within 1 hour,while DR occurs from 2 to 36 hours after drug administration.The IR have a validated method for the in vitro diagnosis,such as the basophil activation test(BAT).For the DR there is a lack of reliable diagnostic methods.Only lymphocyte transformation test(LTT)seems able to detect DR,but it requires 6-7 days of cells incubation and the use of radioactive isotopes to detect T cells activation.The basis of our method(LAT)is very similar to BAT;it requires an incubation of WBC at 37°C for 12-16 hours with drug molecules and a calcium/brefeldin A enriched buffer.Subsequently cells were fixed,permeabilized and labeled by fluorescent mAbs in order to detect surface CD4/CD69 and intracellular IL4 expression.Finally cells were analyzed by flow cytometry to detect activated T cells.In blood samples from patients with delayed sensitization to aminopenicillins(AMP)suspect it was performed LAT.Any subjects referred immediate reaction,others referred delayed reactions to AMP.To evaluate the specificity of LAT we analyzed samples from:6 healthy subjects,12 allergic subjects not sensitized to AMP,7 subjects referring IR to AMP,and 8 allergic subjects referring DR to AMP.Positivity were established calculating ratio between percentage of CD69-IL4 positive T cells in stimulated samples and CD69-IL4 positive cells in unstimulated samples, defined as Stimulation Index(SI).When SI≥2 we consider the test as positive.We observed that healthy subjects,allergic subjects(to other molecules)and subjects with immediate reactions to AMP showed no positivity to LAT while all the 8 subjects referring delayed reactions to AMP showed positivity signals to LAT,of which 5 by CD69, 2 by IL-4 and 1 by CD69/IL-4. We reduced incubation time to 12-16 hours, and simplified test by flow cytometry instead of radioactive methods,thus making LAT vs. LTT more simple and safe to use.These data evidence that our test is more specific,and have a good sensitivity.Despite the small samples,our results are very promising and could be the right way for the development of a reliable in vitro diagnostic protocol for delayed adverse drug reactions as useful support for the prevention of adverse reactions.
A new flow cytometry method for detection of delayed drug reaction to aminopenicillins by in vitro Lymphocyte activation test (LAT)
CARUSO, MASSIMO;Emma R;POLOSA, Riccardo
2013-01-01
Abstract
The adverse "allergic" drug reaction can be distinguished in immediate(IR)and delayed type(DR),based on the time between the drug administration and the onset of the reaction.IR occurs within 1 hour,while DR occurs from 2 to 36 hours after drug administration.The IR have a validated method for the in vitro diagnosis,such as the basophil activation test(BAT).For the DR there is a lack of reliable diagnostic methods.Only lymphocyte transformation test(LTT)seems able to detect DR,but it requires 6-7 days of cells incubation and the use of radioactive isotopes to detect T cells activation.The basis of our method(LAT)is very similar to BAT;it requires an incubation of WBC at 37°C for 12-16 hours with drug molecules and a calcium/brefeldin A enriched buffer.Subsequently cells were fixed,permeabilized and labeled by fluorescent mAbs in order to detect surface CD4/CD69 and intracellular IL4 expression.Finally cells were analyzed by flow cytometry to detect activated T cells.In blood samples from patients with delayed sensitization to aminopenicillins(AMP)suspect it was performed LAT.Any subjects referred immediate reaction,others referred delayed reactions to AMP.To evaluate the specificity of LAT we analyzed samples from:6 healthy subjects,12 allergic subjects not sensitized to AMP,7 subjects referring IR to AMP,and 8 allergic subjects referring DR to AMP.Positivity were established calculating ratio between percentage of CD69-IL4 positive T cells in stimulated samples and CD69-IL4 positive cells in unstimulated samples, defined as Stimulation Index(SI).When SI≥2 we consider the test as positive.We observed that healthy subjects,allergic subjects(to other molecules)and subjects with immediate reactions to AMP showed no positivity to LAT while all the 8 subjects referring delayed reactions to AMP showed positivity signals to LAT,of which 5 by CD69, 2 by IL-4 and 1 by CD69/IL-4. We reduced incubation time to 12-16 hours, and simplified test by flow cytometry instead of radioactive methods,thus making LAT vs. LTT more simple and safe to use.These data evidence that our test is more specific,and have a good sensitivity.Despite the small samples,our results are very promising and could be the right way for the development of a reliable in vitro diagnostic protocol for delayed adverse drug reactions as useful support for the prevention of adverse reactions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
