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The susceptibility to infections was recorded in 13 patients with I3 thalassaemia major (T.P.). The following parameters were also investigated in their polymorphonuclear neutrophils (PMN): nitro blue tetrazolium (NBT) reduction, heated yeast and Escherichia coli phagocytosis, Escherichia coli killing and myeloperoxydase activity. These results were compared to those obtained in healthy controls (H.C.). The Perk's reaction was performed on PMN and graded according to a scoring system with the aim of quantifying the iron intoxication of PMN. Phagocytosis and Perk's reaction of PMN from H.C. were also studied after 20 h of incubation with thalassaemic serum. 6 T.P. out of 13 developed septicaemia during their lifetime and in all 9 septicaemic episodes were noted. Phagocytosis was greatly impaired, disclosing both cellular and serum abnormalities. The mean percentage of Perk's positive PMN was 13% in T.P., contrasting with the constant negative reaction in H.C. The incubation of PMN from H.C. with serum from T.P. induced the simultaneous appearance of a phagocytosis defect and of a positive Perl's reaction. It was concluded that in I3 thalassaemia major the phagocytosis of PMN was altered due to a combination of serum and cellular abnormalities and that both may be related to the iron overload.

Background: Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods: FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt ™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on agematched percentile (p5-p95) reference ranges. In parallel, intra-and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results: The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra-and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on

T he QuantiFERON-TB Gold Plus assay (QFT; Qiagen, Hilden, Germany) is a frequently used interferon gamma releasing assay (IGRA) for the diagnosis of latent tuberculosis infections (LTBI) (1, 2). Recently, it became available on Liaison XL (DiaSorin S.p.A., Saluggia, Italy), a fully automated analyzer using chemiluminescense detection within a chemiluminescent immunoassay (CLIA). In this study, we compared this novel method to the commonly used enzyme-linked immunosorbent assay (ELISA) in a low-incidence LTBI setting. Heparin blood samples (n ϭ 92) taken for routine QFT assays were analyzed with both assays on two different sites as follows: after incubation and centrifugation as instructed by the manufacturer, QFT CLIA was performed on-site (Ghent University Hospital, Belgium) on one aliquot of the processed plasma sample, while another aliquot was transported at room temperature to site two (University Hospital of Leuven, Belgium) for QFT ELISA on the BEP III platform (Siemens Healthcare Diagnostics, Eschborn, Germany). Results were interpreted according to the manufacturer's criteria (positivity threshold 0.35). Of the 92 samples, 46 were from males and 42 from females, median age of 45 years (range 1 to 91 years). Four samples were from encoded health care workers sent by the occupational medicine department. Eighty-seven samples (95%) returned concordant results: 12 positive, 71 negative, and 4 indeterminate results. Five samples (5%) were discordant (Table 1), of which four samples resulted in a major discrepancy, i.e., positive versus negative. Clinical information on these samples did not bring clarity and follow-up samples were not included within the scope of this comparison. However, the discordant samples were all lowpositive results which, when applying a suitable additional range, could be classified as borderline. The use of a borderline range remains subjective but is justified, particularly in a low LTBI setting and among patients with low individual risk for tuberculosis (3). The Belgian Lung and Tuberculosis Association (4) recently suggested defining borderline results as between 0.35 and 0.7, and recommended retesting of borderline samples. As this range has been defined based on QFT ELISAs, extrapolation to other systems might not be justifiable. In this study, CLIA returned significantly higher values for TB1/TB2 tubes than ELISA (Fig. 1). This quantitative difference is possibly due to preanalytics, where QFT CLIA was performed on-site while QFT ELISA required transport to site two, causing a delay of at least one day and leading to possible degradation of interferon gamma. However, in 2 of 5 discordant samples, higher levels (positive results) were measured in the QFT ELISA, suggesting this explanation might fall short. Another possibility is that the difference is intrinsic to the detection method.

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