Geneviève Servais

Simple quantitative haemolytic microassay for determination of complement alternative pathway activation (AP50).

We describe here a simple, reliable and quantitative method to measure the alternative pathway (AP) dependent mode of complement activation for the lysis of rabbit erythrocytes. In the test the reciprocal of the plasma volume needed to destroy 50% of available rabbit erythrocytes is defined as the functional measure of this activity (AP50). The test was found to be highly reproducible both within and between assays with a coefficient of variation which was less than 5%. Sensitivity was also shown to be satisfactory and all of the plasma samples from the healthy blood donors which were tested could be assayed with precision. The specificity of the AP50 assay for AP complement activation was verified by the fact that a C4 affinity-depleted plasma gave an AP50 value within the normal range (52.8 U/ml) while a similar aliquot of the same plasma, affinity-depleted of factor B, gave an undetectable AP50 value (less than 10 U/ml). Furthermore, a sample was unable to lyse the target cells when heated to 50 degrees C or 56 degrees C, treatments which are known to destroy factor B and total haemolytic complement, respectively. To ensure inhibition of the classical pathway of activation. EGTA and MgCl2 were added during the assay. An advantage of this assay is that it is possible, using microplates, multipipettes and a spectrophotometer coupled to a computer, to semi-automate the procedure.

Evidence of autoantibodies to cell membrane associated DNA (cultured lymphocytes): a new specific marker for rapid identification of systemic lupus erythematosus

OBJECTIVE Autoantibodies to cell membrane associated DNA are described in systemic lupus erythematosus (SLE). The specificity of these antibodies differ from antibodies to nuclear DNA. METHODS Using indirect immunofluorescence, a specific IgG was detected giving a characteristic pattern of continuous peripheral membrane fluorescence on cultured B-lymphocytes. RESULTS This pattern was observed in 53 of 80 serum samples of SLE patients but absent in the serum samples of the control populations: 15 rheumatoid arthritis, 38 ankylosing spondylarthritis, 17 non-inflammatory osteopenic patients, and 224 blood donors. In 34 Sjögren syndrome’s patients one only showed a positive test. The cmDNA specificity of these antibodies was confirmed by pattern extinction with DNAse but not RNase or protease pre-treatment of the cells. IgG to cmDNA, separated by absorption/elution from purified cmDNA immobilised on DEAE-nitrocellulose reproduced the immunofluorescence pattern pictures. Extensive serum depletion of anti-double strand or single strand DNA antibodies by absorption to cellulose bound ds- or ss-DNA affected marginally the pericellular fluorescence revealing some minor cross reactivity with nuclear DNA. Moreover, in SLE patients without detectable antibody to ds-DNA, pericellular fluorescence could be visible. CONCLUSION This novel rapid immunofluorescence method may serve as an identification test of SLE patients. Given its positive (97.1%) and negative (92.9%) predictive value, sensitivity (66%) and specificity (99.5%), it improves on other diagnostic tests such as the detection of antibodies to Sm.

Current practices in antinuclear antibody testing: results from the Belgian External Quality Assessment Scheme.

Abstract Background: This study aimed to assess the state-of-the-art of antinuclear antibody (ANA) testing as practiced in the Belgian and Luxembourg laboratories, using the results obtained in the Belgian National External Quality Assessment Scheme from 2000 to 2005. Methods: During this period, nine samples with different specificities were sent for analysis. Participants were surveyed for methodology used and were asked to report staining pattern and titer of ANAs. In 2002, an attempt was made to improve the comparability of quantitative ANA results by the provision of a commercial reference material and to relate observed differences to methodology. Results: With one exception, all participants employed a microscope-based indirect immunofluorescence assay with human epithelial cell line 2 cells. Most laboratories were accurate in describing the pattern. The percentage of unacceptable answers was greater for samples with borderline levels of antibody and for samples showing a cytoplasmic pattern. An improvement in the detection of anticentromere antibodies was observed. For all samples, a wide range of titers was reported. The provision of the secondary reference preparation led to improved inter-laboratory concordance. Comparison of methodology variables revealed a correlation between unstandardized titers and the power of the lamp of the microscope and the use of a dark room. Conclusions: The EQAS results presented in this work provide valuable insights into the state of the art of ANA testing as practiced in the Belgian and Luxembourg Laboratories and illustrate the important value of a national EQAS for ANA testing as a tool to improve performance and interlaboratory comparability of laboratory results. Clin Chem Lab Med 2009;47:102–8.

Anti DNA antibodies are not restricted to a specific pattern of fluorescence on HEp2 cells

Abstract Background: Antinuclear autoantibody determination relies on an initial screening step using immunofluorescence on HEp2 cells. This may be followed by anti-deoxyribonucleic acid (DNA) determination, if the fluorescence of nuclei is homogeneous. We assessed the validity of a restricted algorithm and compared this to a more comprehensive algorithm that accepted any nuclear pattern as a positive indicator. Methods: Our retrospective study analyzed routine results for antinuclear antibodies (ANA) and their anti-DNA identification [double stranded nuclear DNA (ds-DNA), membrane associated DNA (mDNA), nucleosomes] for 58 systemic lupus erythematosus (SLE) patients (690 sera). We included 158 patients with systemic or organ-specific autoimmune diseases (888 sera), 44 with viral disease (88 sera), 34 cancer patients (89 sera) and 111 patients with inflammation that served as controls (122 sera) for a total of 1187 samples. Results: 1) Anti DNA antibodies are not associated only with a homogeneous pattern, but can also be seen with a speckled or nucleolar pattern. 2) The observed pattern is typical for a particular patient rather than for a specific pathology. 3) A homogeneous pattern does not necessarily indicate SLE, nor does the presence of circulating anti DNA antibodies. 4) Determination of various specificities of anti DNA antibodies, whatever the immunofluorescent pattern, increases the sensitivity and specificity for SLE. Conclusions: If diagnosis is based exclusively on a homogenous pattern, preselection would have missed identification of SLE as high levels of anti DNA antibodies were also associated with speckled or nucleolar pattern. Clin Chem Lab Med 2009;47:543–9.

In vivo and in vitro use of plasma membrane DNA in the detection and follow-up of systemic lupus erythematosus

Dear Editor, Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease in which the immune system attacks the patient’s tissues, resulting in inflammation and damage (Tan et al., 1982). SLE is associated with the presence of abnormally elevated titers of a variety of antibodies, including antibodies to doublestranded (ds) DNA, in the serum (Egner, 2000). The presence of anti-dsDNA antibodies in abnormal titers has become the serological hallmark of the disease, and it is widely acknowledged that deposition of those autoantibodies in the form of immune complexes incites the inflammation and tissues damage that are characteristics of SLE (Riboldi et al., 2005). Yet the perfect method for detecting anti-dsDNA remains controversial, whether regarding the antigen source or the technique used (Egner, 2000; Riboldi et al., 2005; McCloskey et al., 2010). Here we describe a simplified method for the isolation and purification of DNA associated with the plasma membrane of cultured human lymphocytes (Wil2), hereafter designated ‘pmDNA’ that is shown, by electrophoretic mobility shift assay (EMSA), to form a complex with IgG isolated from SLE patients’ sera but not with IgG isolated from healthy individuals’ sera. We also show that Wil2 pmDNA is a choice antigen for the detection of anti-dsDNA in the serum of SLE patients by an indirect immunofluorescence in vivo assay. Wil2 lymphocytes incubated with untreated SLE patients’ sera and then with fluorescein-conjugated goat anti-human IgG exhibited a ring-like peripheral membrane fluorescence (Figure 1A, a) while no fluorescence was observed with healthy sera (Figure 1A, b). In addition to cells exhibiting a continuous distribution of the fluorescence, others exhibiting patching or capping were also seen with SLE sera (Figure 1A, c–e). This reflected a ligand-induced movement of lymphocyte membrane macromolecules (Cohen and Gilbertsen, 1975; Pavan et al., 1990), indicating that the fluorescent anti-human IgG bound to an IgG–plasma-membraneantigen complex. Fluorescence was also observed when Wil2 cells were pre-treated with RNase (Figure 1A, f), pronase E (Figure 1A, g), or phospholipase C (Figure 1A, h), but not with DNase (Figure 1A, i), indicating that the plasma membrane antigen that bound the SLE patients’ IgG was DNA. Sera of patients with other autoimmune diseases were also submitted to the indirect immunofluorescence assay using Wil2 lymphocytes. While at dilution 1/2, fluorescence was observed with 4 of 15 Sjögren’s syndrome sera, 2 of 10 spondylarthritis sera, and 5 of 15 rheumatoid arthritis sera, no fluorescence was observed at dilution 1/40, regardless of the serum’s origin. In contrast, fluorescence was observed with SLE sera, regardless of the dilution. When all sera were tested for the presence of anti-dsDNA using a routine enzyme-linked immunosorbent assay (ELISA) with calf-thymus DNA as antigen, results showed that the indirect immunofluorescence test described here was more sensitive and more specific in detecting anti-dsDNA IgG in the sera of SLE patients than the routine ELISA. Previous investigations indicated that the use of cultured B-lymphocytes (Wil2) in an indirect immunofluorescence test allowed for the detection of specific IgG in SLE patients with a higher specificity and sensitivity than other assays (Keyhani et al., 1995), but the adequacy of the test for anti-dsDNA detection was not unequivocally shown. IgG fractions were isolated from the sera of healthy individuals (Figure 1B, a) and untreated SLE patients (Figure 1B, a, inset) by affinity chromatography on protein G-Sepharose. The second peak, corresponding to IgG (see Supplementary data), exhibited two bands (identified, respectively, as the heavy and light chains of immunoglobulins) after SDS–polyacrylamide gel electrophoresis (Figure 1B, b). The pmDNA isolated from Wil2 plasma membrane (see Supplementary data) revealed a single band upon electrophoresis in agarose gel. The band was eluted from the gel so as to remove residual RNA (Figure 1C, a), giving a pmDNA 17000 bp-long, free of RNA or protein contamination (Figure 1C, b): the band corresponding to the purified pmDNA was no longer detectable after pmDNA treatment with DNase (lane 2), but was unaltered after pmDNA treatment with RNase (lane 3) or pronase E (lane 4). pmDNA mobility in agarose gel was remarkably reduced after incubation with IgG from SLE sera (Figure 1D, a and c) but remained unaffected after incubation with IgG from control sera (Figure 1D, b). The reduction in pmDNA mobility was dose dependent, with complete inhibition in the presence of 10 mg SLE IgG and various extents of inhibition, depending on the patient, at lower amounts of SLE IgG (Figure 1D, a, lane 4 vs. c, lane 3). These results indicated that a complex was formed between pmDNA and IgG isolated from SLE sera but that no complex was formed between pmDNA and control IgG, confirming the immunofluorescence test results. Formation of an IgG–pmDNA complex was also examined by co-incubating pmDNA with SLE IgG and pronase (pronase alone does not alter pmDNA migration as shown in Figure 1C, b). In the absence of pronase, an IgG–pmDNA complex was 200 | Journal of Molecular Cell Biology (2011), 3, 200–201 doi:10.1093/jmcb/mjq051 Published online April 21, 2011