Performance characteristics of a new automated method for measurement of anti-cyclic citrullinated peptide.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory auto-immune disease affecting approximately 1%-2% of the population worldwide. RA is a potentially crippling disease since it results in malformation of the joints. RA is mostly diagnosed based on clinical manifestations but serological tests against autoantibodies, such as rheumatoid factor and anti-cyclic citrullinated peptides (aCCP), are available. The presence of aCCP antibodies is strongly associated with a more severe, destructive disease course. Recently, a new test for the measurement of aCCP antibodies on the IMMULITE 2000(XPi) platform was developed by Siemens Healthcare. In this study we investigated the performance characteristics of this new aCCP test in four different hospital laboratories and compared the new test with three different commercially available platforms. METHODS: Samples were collected from patients presented to the hospital for aCCP measurement. Serum aCCP levels were determined by aCCP (Ig)G assay for IMMULITE 2000(XPi) systems (Siemens Healthcare), ImmunoScan RA enzyme-linked immunosorbent assay (ELISA) test (Eurodiagnostica), Immunocap 250 (Thermofisher) or aCCP IgG assay on the Modular system (Roche Diagnostics). The evaluation protocol consisted of within-run imprecision (20 sequential runs), between-run imprecision (16 workdays), comparison of serum and plasma measurement and method comparison. RESULTS: The within-run imprecision (n=20) for aCCP IgG assay on three different IMMULITE 2000(XPi) systems ranged from 3.0% to 6.9% at levels 3.2-171.2 U/mL. Between-run imprecision (n=16 days) ranged from 5.2% to 11% at levels of 3.2-106.9 U/mL. Method comparison showed good correlation when samples were measured on two different Immulite analyzers in two different hospital laboratories [0.21+0.96x (n=40)]. Method comparison of the IMMULITE 2000(XPi) aCCP test with aCCP on Immunoscan RA ELISA (n=112), Immunocap 250 (n=105) and the Modular system (n=289) resulted in a concordance of 90.2%, 93.3% and 94.8%, respectively. Correlation of serum versus heparin samples showed a correlation of 0.12+1.08x for the Immulite 2000(XPi) test. CONCLUSIONS: The aCCP assay on the IMMULITE 2000(XPi) has good performance characteristics and shows high level of concordance with the aCCP test on Immunoscan RA ELISA test, Immunocap 250 and the Modular systems.

Diagnostic efficiency of the Sysmex XN WPC channel for the reduction of blood smears.

BACKGROUND: Several small studies showed that the WPC (white precursor cell) channel in the Sysmex haematology analyser for leukocyte differentiation analysis leads to a significant reduction of microscopic blood smears. We determined the added value of the WPC channel for blood smear reduction in a large teaching hospital and whether this reduction was cost-effective and save. METHODS: Retrospectively, for 7850 leukocyte differentiation orders the percentage of samples resulting in a WPC analysis and the outcomes of the WPC analysis were analysed and compared with the blood smear results. RESULTS: WPC analysis resulted in a 12% reduction of blood smears, which is much lower than observed in other studies. This means 3-4bloodsmears/day less of a total of 28smears/day at the expense of missing 14 patient samples with pathology (blasts or abnormal lymphocytes) in a 9weeks period. The estimated total costs of WPC analysis per year was more than the reduction in costs due to less blood smear reviews. CONCLUSIONS: In a large teaching hospital, the small reduction in blood smears by the WPC channel does not outweigh the costs and the slight reduction in sensitivity for pathology.

Langerhans cells are negative regulators of the anti-Leishmania response.

Migratory skin dendritic cells (DCs) are thought to play an important role in priming T cell immune responses against Leishmania major, but DC subtypes responsible for the induction of protective immunity against this pathogen are still controversial. In this study, we analyzed the role of Langerin(+) skin-derived DCs in the Leishmania model using inducible in vivo cell ablation. After physiologically relevant low-dose infection with L. major (1,000 parasites), mice depleted of all Langerin(+) DCs developed significantly smaller ear lesions with decreased parasite loads and a reduced number of CD4(+) Foxp3(+) regulatory T cells (T reg cells) as compared with controls. This was accompanied by increased interferon γ production in lymph nodes in the absence of Langerin(+) DCs. Moreover, selective depletion of Langerhans cells (LCs) demonstrated that the absence of LCs, and not Langerin(+) dermal DC, was responsible for the reduced T reg cell immigration and the enhanced Th1 response, resulting in attenuated disease. Our data reveal a unique and novel suppressive role for epidermal LCs in L. major infection by driving the expansion of T reg cells. A better understanding of the various roles of different DC subsets in cutaneous leishmaniasis will improve the development of a potent therapeutic/prophylactic vaccine.

Functional redundancy of Langerhans cells and Langerin+ dermal dendritic cells in contact hypersensitivity.

The relative roles of Langerhans cells (LC), dermal dendritic cells (DC), and, in particular, the recently discovered Langerin(+) dermal DC subset in the induction and control of contact hypersensitivity (CHS) responses remain controversial. Using an inducible mouse model, in which LC and other Langerin(+) DC can be depleted by injection of diphtheria toxin, we previously reported impaired transport of topically applied antigen to draining lymph nodes and reduced CHS in the absence of all Langerin(+) skin DC. In this study, we demonstrate that mice with a selective depletion of LC exhibit attenuated CHS only upon sensitization with a low hapten dose but not with a high hapten dose. In contrast, when painting a higher concentration of hapten onto the skin, which leads to increased antigen dissemination into the dermis, CHS is still diminished in mice lacking all Langerin(+) skin DC. Taken together, these data suggest that the magnitude of a CHS reaction depends on the number of skin DC, which have access to the hapten, rather than on the presence or absence of a particular skin DC population. LC and (Langerin(+)) dermal DC thus seem to have a redundant function in regulating CHS.

Langerhans cells are required for UVR-induced immunosuppression.

Painting of haptens onto UVR-exposed skin does not result in sensitization but induces regulatory T cells (Treg). This was explained by UVR-mediated depletion of Langerhans cells (LCs). Furthermore, migration of UVR-damaged but still viable LCs into lymph nodes appears to be essential to induce Treg. Accordingly, the steroid mometasone, which kills LCs, inhibited sensitization but did not induce Treg. In Langerin-diphtheria toxin receptor knock-in (DTR) mice, LCs can be depleted by injection of diphtheria toxin (DT). LC-depleted mice could be sensitized though less pronounced than wild-type mice, but sensitization was not suppressed by UVR. Similarly, Treg did not develop. Langerin is not only expressed in LCs but also in some dermal dendritic cells (dDCs). Langerin-positive dDCs repopulate within 10 days after depletion, whereas LCs are still absent. Langerin-DTR mice treated with DT 10 days before UVR and sensitization were still resistant to UVR-induced inhibition of contact hypersensitivity (CHS). Similarly, Treg did not arise. As in this setting only LCs but not Langerin-positive dDCs are absent, LCs appear to be essential for both the suppression of CHS and the induction of Treg by UVR. This supports the concept that LCs are more important for the downregulation than the induction of immune responses in the skin.

Langerhans cells are required for efficient presentation of topically applied hapten to T cells.

Dendritic cells (DC) play a pivotal role in the control of T cell immunity due to their ability to stimulate naive T cells and direct effector function. Murine and human DC are composed of a number of phenotypically, and probably developmentally, distinct subsets, which may play unique roles in the initiation and regulation of T cell responses. The skin is populated by at least two subsets of DC: Langerhans cells (LC), which form a contiguous network throughout the epidermis, and dermal DC. LC have classically been thought vital to initiate T cell responses to cutaneous Ags. However, recent data have highlighted the importance of dermal DC in cutaneous immunity, and the requirement for LC has become unclear. To define the relative roles of LC and dermal DC, we and others generated mouse models in which LC were specifically depleted in vivo. Unexpectedly, these studies yielded conflicting data as to the role of LC in cutaneous contact hypersensitivity (CHS). Extending our initial finding, we demonstrate that topical Ag is inefficiently transported to draining lymph nodes in the absence of LC, resulting in suboptimal priming of T cells and reduced CHS. However, dermal DC may also prime cutaneous T cell responses, suggesting redundancy between the two different skin DC subsets in this model.

Proinflammatory mediator-induced reversal of CD4+,CD25+ regulatory T cell-mediated suppression in rheumatoid arthritis.

OBJECTIVE: We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg-mediated suppression. METHODS: Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme-linked immunosorbent assay. Magnetically sorted CD4+,CD25- and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti-CD3 monoclonal antibody (mAb) and autologous antigen-presenting cells, in the absence or presence of anti-CD28 mAb or the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), or IL-7. RESULTS: Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB-derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti-CD28 mAb to cocultures of CD4+,CD25- and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg-mediated suppression in both PB and SF. Furthermore, IL-7 and, to a limited extent, TNFalpha, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg-mediated suppression. In contrast, IL-6 did not influence Treg-mediated suppression. CONCLUSION: Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.