Nonradioactive Techniques for Measurement of In Vitro T-Cell Proliferation: Alternatives to the [(3)H]Thymidine Incorporation Assay

T-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [(3)H]thymidine ([(3)H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [(3)H]TdR assay as a “gold standard.” As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2′-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [(3)H]TdR incorporation in both study groups (HIV-1 positives, r = 0.96; HIV-1 negatives, r = 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [(3)H]TdR incorporation, both in HIV-1-positive (r = 0.96) and HIV-1-negative (r = 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T-cell proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers

Autoantibody standardization in The Netherlands.

Item does not contain fulltextSeveral initiatives have been undertaken, independent of the European Autoantibody Standardization Initiative (EASI), to standardize autoantibodies in The Netherlands. The Dutch EASI team has made an inventory of which initiatives on autoantibody standardization are already available and what future plans for autoantibody standardization exist. This inventory will subsequently be used to define what may be addressed by the Dutch EASI team. The Diagnostic Compass, initiated by the association of Dutch health insurance companies, describes methods and relevance of laboratory tests, including autoantibody tests. Recently, this initiative has been taken over by an independent publisher. There is also a national organization involved in developing guidelines in medicine, including guidelines for autoantibody testing. In addition, there is a national foundation for quality assessment in clinical laboratories (SKML). The quality assessment includes a wide array of autoantibodies. Samples are collected and thoroughly investigated by reference laboratories. Interpretation of results and advice to clinicians are part of the program. Feedback on the results of this proficiency testing is given in reports and during meetings to discuss trends, technical issues, and new developments. The last initiative that we discuss is the foundation Referentie Laboratorium Reuma Serologie (RELARES), which was founded to standardize serology in rheumatic diseases by preparing standard sera. Recently, RELARES has been combined with SKML. A new SKML working group, Standardization Autoimmune Serology, has been initiated to continue the work of RELARES. When comparing the already available Dutch initiatives to the international EASI goals, there appears to be a lack of harmonization in testing algorithms, and this issue is the most important topic to be addressed in the near future

Comparison of Commercially Available Assays for Infliximab Concentrations and Antibodies to Infliximab With Assays Developed at Janssen and Used in Clinical Studies of Remicade (R) : Iinfliximab in IBD Patients

status: publishe

Expansion of CMV-specific CD8+CD45RA+CD27- T cells in B-cell chronic lymphocytic leukemia

In patients with B-cell chronic lymphocytic leukemia (B-CLL), the absolute number of T cells is increased. Although it has been suggested that these T cells might be tumor specific, concrete evidence for this hypothesis is lacking. We performed a detailed immunophenotypic analysis of the T-cell compartment in the peripheral blood of 28 patients with B-CLL (Rai 0, n = 12; Rai I-II, n = 10; Rai III-IV, n = 6) and 12 healthy age-matched controls and measured the ability of these patients to mount specific immune responses. In all Rai stages a significant increase in the absolute numbers of CD3+ cells was observed. Whereas the number of CD4+ cells was not different from controls, patients with B-CLL showed significantly increased relative and absolute numbers of CD8+ cells, which exhibited a CD45RA+CD27- cytotoxic phenotype. Analysis of specific immune responses with tetrameric cytomegalovirus (CMV)-peptide complexes showed that patients with B-CLL had significantly increased numbers of tetramer-binding CMV-specific CD8+ T cells. The rise in the total number of CD8+ cytotoxic T cells was evident only in CMV-seropositive B-CLL patients. Thus, our data suggest that in patients with B-CLL the composition of T cells is shifted toward a CD8+ cytotoxic cell type in an effort to control infections with persistent viruses such as CMV. Moreover, they offer an explanation for the high incidence of CMV reactivation in CLL patients treated with T cell-depleting agents, such as the monoclonal antibody (mAb) alemtuzumab (Campath; alpha-CD52 mAb). Furthermore, because in CMV-seronegative patients no increase in cytotoxic CD8+ T cells is found, our studies do not support the hypothesis that tumor-specific T cells account for T-cell expansion in B-CL

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-1γ, anti-NXP-2 and anti-SAE. Moreover, anti-MDA-5 is associated with amyopathic myositis and interstitial lung disease and anti-TIF-1γ 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.status: publishe

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

International Consensus on ANA Patterns (ICAP):inbedding in het Nederlandse taalgebied

In het kader van harmonisatie van de rapportage van autoimmuundiagnostiek is er sinds enkele jaren internationale consensus over de nomenclatuur en definitie van HEp-2-patronen die met behulp van indirecte immuunfluorescentie (IIF) te onderscheiden zijn. Deze test wordt gebruikt om anticellulaire antistoffen aan te tonen die veelal gerapporteerd worden als antinucleaire antistoffen (ANA) en een belangrijke rol spelen in de diagnostiek van systemische autoimmuunziekten. In een nauwe samenwerking tussen het Belgische en Nederlandse EASI-team is de consensus beschikbaar gemaakt voor het Nederlandse taalgebied. Uitgangspunt is dat de Nederlandstalige benamingen van de verschillende HEp-2-patronen breed ingevoerd gaan worden in klinische laboratoria in Nederland en Vlaanderen. Inleidin