DIHYDRORHODAMINE FLOW CYTOMETRIC TEST DIHYDRORHODAMINE FLOW CYTOMETRIC TEST -APPLICATION IN TOLL-IL1R DEFICIENCIES-
- Alexandru D. Vlagea, Spain
Abstract
Background and Aims
Evaluation of phagocyte respiratory oxidative burst using dihydrorhodamine test (DHR-test) allows a fast diagnosis of Chronic Granulomatous Disease(CGD). DHR-test often employs phorbol myristate acetate (PMA) and E.coli as standard stimuli of NF-kB signaling pathway. PMA signals through Protein Kinase C (PKC), whereas E.coli triggeres Toll-like Receptors (TLRs)-mediated activation and probably other pattern recognition receptors. Patients with Toll-IL-1R(TIR) deficiencies and CGD usually suffer pyogenic infections. However, the specific functional tests for TIR deficiencies are usually available only in specialized laboratories. Herein we aimed to assess the capacity of DHR test to screen for TIR deficiencies in a diagnostic setting.
Methods
Four patients with a definitive diagnosis of TIR deficiency (two IRAK-4- and two MyD88-deficient), 3 healthy family members with heterozygous IRAK-4 genotypes (HFM), and 10 healthy control subjects (HC) were analyzed by DHR-test with PMA and E.coli.
Results
Gating on neutrophils, the Stimulation Index (SI) = (MFI-DHRstimulus/MFI-DHRnegative control) was calculated. All enrolled individuals showed normal results (SIPMA>30) in DHR-test with PMA.TIR-deficient patients showed a marked reduction of DHR-test responses upon E. coli stimulation (mean SIE.coli:45.3) compared with healthy controls (mean SIE.coli: 100.56) and HFM (mean SIE.coli:59.8). When the ratio between SIE.Coli/SIPMA was calculated, all TIR-deficient patients showed ratios <0.5 while 9/10 controls and all the heterozygous relatives displayed ratios >0.5 (Figure 1)
Conclusions
TIR-deficient patients show a specific impairment of E.coli DHR-test as opposed to PMA responses. This dissociation is not observed in CGD patients (both responses equally affected), nor in HFM. Therefore, DHR test using both PMA and E.coli could reveal TIR deficiency.