Propylthioiracil induced ANCA-associated vasculitis in a 14 year-old girl

Background: Antineutrophil cytoplasmic antibodies (ANCAs) are the serologic hallmark of ANCA-associated primary small-vessel vasculitides (AAVs), but these antibodies have also been described in other autoimmune diseases such as inflammatory bowel diseases. Furthermore, different drugs are linked to the induction of ANCA, including propylthiouracil (PTU). However progression into clinical overt vasculitis is less common. Case-diagnosis/treatment: We describe the case of a young girl with Graves' disease presenting with fatigue, fever, episcleritis and arthritis. The unexpected double myeloperoxidase/proteinase 3-ANCA positivity triggered a multidisciplinary diagnostic work-up and resulted in the diagnosis of a clinically overt PTU-induced AAV. After PTU-withdrawal and treatment with high-dose corticosteroids, a favorable clinical and biochemical evolution was obtained. Conclusions: The use of PTU in the management of hyperthyroidism is not considered first-line treatment in Europe and is even less commonly used in children. Nevertheless, pediatricians should be aware of the possibility of PTU-induced AAV, especially in the presence of multiple ANCA reactivities. Therefore, the use of this drug should be weighed carefully in children

Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (approximate to transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies. EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 degrees C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for < 24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes. Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population

Optimization and testing of dried antibody tube: The EuroFlow LST and PIDOT tubes as examples

Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orientation tube (PIDOT). Both tubes were evaluated on normal and/or on patient samples, comparing the mean fluorescence intensity of specific lymphocyte populations. Our data show that the dried tubes and liquid counterparts give highly comparable staining results, particularly when analyzed in multidimensional plots. In addition, the use of dried tubes may result in a reduced staining variability between different samples and thereby contributes to the generation of more robust data. Therefore, by using ready-to-use reagents in a dried single test tube format, the laboratory efficiency and quality will be improved

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 1

Autoantibodies in idiopathic inflammatory myopathies:Clinical associations and laboratory evaluation by mono- and multispecific immunoassays

Idiopathic inflammatory myopathies (IIM) are a group of diseases characterized by immune-mediated muscular lesions that may be associated with extra-muscular manifestations involving skin, lungs, heart or joints. Four main groups of IIM can be distinguished: dermatomyositis (DM), overlap myositis including mainly anti-synthetase syndrome (ASS), immune mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM). Myositis-specific autoantibodies (MSA) are increasingly recognized as valuable tools for diagnosis, classification and prognosis of IIM. For example, ASS is associated with anti-aminoacyl tRNA synthetase antibodies (anti-Jo-1, PL-7, PL-12,...), IMNM with anti-SRP and anti-HMGCR; IBM with anti-cytosolic 5'nucleotidase 1A (cN1A), and DM with anti-Mi-2, anti-MDA-5, anti-TIF-1y, anti-NXP-2 and anti-SAE. Moreover, anti-MDA-5 is associated with amyopathic myositis and interstitial lung disease and anti-TIF-1y and anti-NXP-2 with juvenile DM as well as malignancy in patients > 40 years. Most MSA have initially been discovered by immunoprecipitation. In routine laboratories, however, MSA are screened for by indirect immunofluorescence and identified by (automated) monospecific immunoassays or by multispecific immunoassays (mainly line/dot immunoassays). Validation of these (multispecific) assays is a challenge as the antibodies are rare and the assays diverse. In this review, we give an overview of the (clinical) performance characteristics of monospecific assays as well as of multispecific assays for detection of MSA. Although most assays are clinically useful, there are differences between techniques and between manufacturers. We discuss that efforts are needed to harmonize and standardize detection of MSA