Measurement of immune complexes is not useful in routine clinical practice

Transcription

1 Review Article Ann Clin Biochem 2000; 37: 253±261 Measurement of immune complexes is not useful in routine clinical practice R J Lock and D J Unsworth From the Department of Immunology, Southmead Hospital, Bristol BS10 5NB, UK The vogue for the determination of immune complexes (ICs) in the clinical setting has now passed, as can be seen from the fact that only two of the regional immunology reference laboratories in the UK still offer a service. This is partly due to the lack of speci city seen in the assays available, which, apart from minor modi cations, have remained essentially unchanged. They have not lived up to their initial promise and are unlikely to be offered by service laboratories in the future. In this review we consider when these assays may still have a role and which might be the most useful in a particular clinical setting. An IC is formed when an antibody reacts speci cally with its corresponding antigen. The size of the complex formed is dependent on many factors, including the ratio of antibody to antigen, the valency of the antigen and the avidity of the antibody±antigen reaction. IC formation is an integral part of the normal immune response to infection and usually takes place without adverse pathological consequences. ICs are captured by speci c receptors, in particular on erythrocytes, and transported to the reticuloendothelial system for processing and removal. The events leading to IC formation and subsequent removal are summarized in Fig. 1. There are, however, various disorders in which ICs play an important role in the disease process. These include some forms of vasculitis (e.g. Henoch±SchoÈ nlein purpura), infectious disorders (e.g. bacterial endocarditis, shunt nephritis) and connective tissue autoimmune disorders [e.g. rheumatoid arthritis (RA), systemic lupus erythematosus (SLE)]. The pathology generally arises from inappropriate clearance or deposition of ICs. Thus, vasculitis This article was commissioned by the Clinical Laboratory Investigations Standing Committee of the Association of Clinical Biochemists. The views expressed are those of the authors. Correspondence: Dr D J Unsworth. joeunsworth@hotmail.com results from IC deposition in vessel walls, nephritis from glomerular deposits and some forms of arthritis from deposition in the joints. In the 1970s and 1980s, recognition of the involvement of IC in disease led to the development of a wide variety of methods for their detection, either in the form of `circulating immune complexes (CIC) in body uids, or deposited IC in biopsy tissue. The latter remains a useful diagnostic tool. DIRECT VISUALIZATION IN BIOPSY TISSUE BY IMMUNOHISTOCHEMISTRY This technique is of genuine clinical worth. Biopsy tissue sections are stained for the presence of either immunoglobulin (Ig), complement components or speci c antigen (e.g. DNA in SLE) using speci c antisera. The antisera may be labelled in one of several ways: immuno- uorescent, immunoenzyme (usually peroxidase or alkaline phosphatase) or for immuno-electron microscopy. This allows direct visualization of the IC and its site of deposition. The presence of immunoglobulin and complement together is assumed to represent local IC (e.g. IgA and C3 in skin biopsy from dermatitis herpetiformis patients). It should be remembered, however, that not all immunoglobulin detected will represent CIC. Note, for example the direct autoantibody binding of IgG to the glomerular basement membrane in Goodpasture s disease. Figure 2 shows an immuno uorescence picture of renal glomerular deposits of ICs in SLE. DETECTION OF CIC IN BODY FLUIDS (USUALLY SERUM) At least 40 different assays for CICs have been described. They vary widely in terms of disease sensitivity, speci city and clinical applicability. Possibly the major area of investigation has been in autoimmune disease, in particular SLE. SLE has been considered the archetypal IC disease. Many of the disease manifestations, including 253

2 254 Lock and Unsworth C1q binding to immune complex Immune complex formation Classical pathway activation Complex bound to erythrocyte via specific receptor Phagocytic cells in liver and spleen remove complexes Key: Antibody Antigen C3b C1q Erythrocyte CR1 receptor FIGURE 1. The formation and clearance of circulating immune complexes. renal disease, arthritis and neurological symptoms, have been attributed to IC, and many of the studies quoted here have attempted to correlate the levels of detectable IC with disease exacerbation. It should be noted in particular that the renal disease in SLE may manifest in several forms (mesangial, membranous, focal or diffuse proliferative) and that these are often simply lumped together as `lupus nephritis. Further, `disease exacerbation may mean a worsening of any aspect of the disease ± renal, cerebral, joint or cutaneous. We will summarize the principles and advantages of some of the more robust of these assays. Broadly, they fall into three groups. First, there are those that utilize physical properties such as size or cryoprecipitability. Second, there are those that utilize biological properties of the IC, such as the ability to bind to speci c receptors on cultured cells (e.g. the Raji cell assay). Third, one can look indirectly for secondary effects of ICs, such as the consumption of complement components. All assays for CIC require good sample collection. Ideally, samples should be collected into a plain glass tube, without clot activator. These should be allowed to clot and then separated at 37 Ê C. Transfer of samples from the patient to the laboratory should be standardized and as rapid as possible. Serum samples should be stored in aliquots at 3 70 Ê C. Repeated freezing and thawing must be avoided as this will aggregate IgG and lead to false positives in many CIC assays.

3 Immune complexes in routine clinical practice 255 FIGURE 2. Immuno uorescence of glomerular deposits of immune complexes in systemic lupus erythematosus (SLE). Staining is with anti-human IgG± uorescein isothiocyanate. Similar patterns are seen when anti-c3 or anti- C1q antibodies are used as indicator. Similar precautions are necessary in the separation of serum for the investigation of cryoglobulinaemia. Failure to follow these principles may lead to loss of the cryoprecipitate during its isolation. In contrast to other investigations for CIC, the serum is cooled to 4 Ê C, rather than frozen at 3 70 Ê C, prior to analysis. In most cases a precipitate will form in the rst 24 h but may take up to 72 h. By de nition, the cryoglobulin should fully redissolve on rewarming to 37 Ê C. Failure to do so suggests the presence of artefactual debris. Unless the possible presence of cryo brinogen is being speci cally investigated, plasma should not be used. The temperature at which the cryoprotein precipitates may be of theoretical interest (`thermal pro ling ). A cryoprotein that precipitates at 22 Ê C is more likely to cause signi cant clinical problems than one precipitating at 4 Ê C. However, in practice, patients are almost invariably symptomatic at presentation and thermal pro ling does not normally help in the diagnosis or management of cryoglobulinaemia. PHYSIOCHEMICAL DETECTION METHODS FOR CICs Cold precipitation/cryoglobulin detection This is a relatively insensitive but nevertheless very useful method for detecting ICs. Complexed immunoglobulins that precipitate in the cold are called cryoglobulins. Not all cryoprecipitable immunoglobulin is attributable to ICs. Brouet et al. established a working classi cation as long ago as In brief, there are three types of cryoglobulins (Table 1). Type I are monoclonal immunoglobulins, usually associated with myeloma or macroglobulinaemia. These are actually immune aggregates rather than ICs, because precipitation does not depend on interaction with an antigen but solely on the innate insolubility of the protein itself. Types II and III are both the result of rheumatoid factor (RF) activity, usually IgM anti-igg. In the case of type II, the RF is monoclonal and in type III it is polyclonal. Thus types II and III are the result of true IC formation in which the antigen is autologous IgG. Cases often arise after infection

4 256 Lock and Unsworth TABLE 1. Classi cation of cryoglobulins First component Rheumatoid factor Second component Major disease associations Approximate proportion of total cases of cryoglobulinaemia (%) Type I, single monoclonal Type II, mixed monoclonal/ polyclonal Type III, polyclonal Monoclonal IgM, IgG (particularly IgG2 or IgG3), IgA (rarely Bence Jones protein) Monoclonal usually IgM No None B-cell malignancy in 90% Yes IgG B-cell malignancy, infection, autoimmunity Polyclonal Yes IgG Infection, autoimmunity After Brouet et al. 1 Published with permission. [e.g. hepatitis C virus (HCV)], or complicating autoimmune disease, especially connective tissue diseases such as SLE. Treatment of the underlying disorder may lead to resolution of the cryoglobulin. Type I and type II cryoglobulins almost invariably cause symptoms. Type III less frequently have a direct role in disease pathology. The presence of a cryoglobulin indicates the need to fully investigate the patient for the presence of lymphoreticular neoplasia. Cryoglobulins contribute to the pathology of the disease, often leading to hyperviscositysyndrome and vasculitis. Other manifestations of IC disorders may also be seen (e.g. glomerulonephritis). The detection of a cryoglobulin should thus prompt the further investigations shown in Table 2. TABLE 2. Further laboratory investigations prompted by the nding of cryoglobulinaemia Investigation Clinical condition sought Full blood count Haematological malignancy Rheumatoid factor (serum and Subtype of cryoglobulinaemia supernatant) Serum electrolytes Renal disease Urine: creatinine clearance, protein Renal disease Complement: C3 and C4 Monitoring therapy Quanti cation of cryoglobulin Monitoring therapy Hepatotropic virus screen HBV, HCV infection Liver function tests HBV=hepatitis B virus; HCV=hepatitis C virus. Quanti cation of the cryoprecipitate may be useful in monitoring therapy (Fig. 3). This may be done either by simple measurement of the precipitate as a percentage of serum volume (the `cryocrit ) or by redissolving the precipitate at 37 Ê C and assaying the protein content. If the cryoglobulin concentration is high, it may also be possible to quantify it by subtracting the amount of supernatant immunoglobulin from the total amount in the original sample. The patient whose data are shown in Fig. 3 had an overlap syndrome that included features of SjoÈ gren s disease and lupus. Acute renal dysfunction led to investigation for and detection of the cryoglobulin, and treatment included prednisolone and cyclophosphamide. Stopping the cyclophosphamide after day 150 led to relapse. Note that the high level of IC led to complement consumption and reversible lowering of the C4 concentration, which therefore provided an alternative marker of disease activity in this patient. This is a pattern we nd quite commonly in patients with cryoglobulinaemia. Analysis of the accumulated data from many studies has shown type II cryoglobulinaemia to be strongly associated with HCV infection (e.g. Miescher et al. 2 ). Agnello et al. reported that anti-hcv antibodies and almost all the viral RNA were concentrated in the cryoprecipitate. 3 Conversely, Hartmann et al. showed that 28/55 (51%) patients with chronic HCV infection had cryoglobulinaemia (mean cryocrit 2 2%). 4 In this study few were typed, but both type II and type III cryoglobulins were found. Similarly, Lunel et al. found 69/127 (54%) patients with HCV hepatitis to have cryoglobulinaemia (22

5 Immune complexes in routine clinical practice 257 FIGURE 3. Serological data on patient with type II cryoglobulinaemia (monoclonal IgM-k, polyclonal IgG, rheumatoid factor-positive) secondary to connective tissue disease. Arrows denote episodes of plasmapheresis. &, cryoglobulin; }, C4. Ig=immunoglobulin. type II, 47 type III). 5 Importantly, Pozzato et al. detected a high prevalence (9/31; 29%) of lowgrade non-hodgkin s lymphoma in HCVassociated type II cryoglobulinaemia. 6 Initial response to interferon therapy in type II cryoglobulinaemia is usually good. However, fewer than 25% of patients achieve long-term remission with standard therapy. 7±9 It should be noted that, although HCV is the predominant disease association in hospital inpatients with type II cryoglobulinaemia, it is not the only cause. An association with hepatitis B virus (HBV) is well known. 10 Critical review 11 has reduced the emphasis of this nding, with cryoglobulinaemia being less frequent in HBV than early studies might suggest. Lunel et al., for example, found cryoglobulins in 15% of 40 patients with HBV. 5 Separation/measurement on the basis of IC molecular weight The polyethylene glycol (PEG) precipitation assay High molecular weight compounds will precipitate ICs from solution by the principle of exclusion. The most popular of these compounds for this purpose is polyethylene glycol (PEG), which is usually added to a nal concentration of 2±3%. The assay, which includes an overnight cold precipitation step, may also detect cryoprotein, even in samples that have been separated at room temperature (personal observation). The PEG precipitation assay is simple in principle, and analysis of the component parts of the precipitate, by enzyme-linked immunosorbent assay (ELISA) or nephelometry, may yield additional speci city. Valentijn et al. applied this approach to SLE patients. 12 Increases in PEG-precipitable IgG or C3 showed a high speci city for disease exacerbation (84% and 99%) but with a low sensitivity (9% and 3%). Further, Coppo et al. did not nd PEG precipitation a useful predictor of disease change in patients with lupus nephritis. 13 The assay is relatively simple to perform and commercial assays are available. It should be noted that monomeric IgG is also precipitated in small amounts, even from normal serum. This interference by monomer is made worse by hypergammaglobulinaemia, 14 which is common in autoimmune disorders (e.g. SjoÈ gren s syndrome). Sucrose density centrifugation Sucrose density centrifugation, with or without subsequent immunochemical analysis of the high

6 258 Lock and Unsworth molecular weight fractions isolated from serum proteins, is very time-consuming and, although useful as a research tool, is not suitable for use in the diagnostic laboratory, where there is a need to process many samples. DETECTION BASED ON BIOLOGICAL PROPERTIES OF CICs Binding of complement added in vitro C1q binding assays C1q is the rst component of the classic pathway of complement. C1q binding assays use the high functional af nity of C1q for conformationally altered immunoglobulin Fc regions, as found in CIC, to distinguish between free and complexed immunoglobulins. C1q binds weakly to monomeric IgG and IgM, but binding is enhanced when these proteins form physical aggregates. C1q-based assays detect only complexes activating the classic complement pathway. C1q binding assays are designed either as uid-phase or solid-phase assays. Fluid-phase assays (C1qFP) generally use 125 I-labelled C1q in the rst stage and PEG to precipitate out the complex, as described by Zubler et al. in The solid-phase C1q binding assay (C1qSP) originally used C1q-coated plastic tubes to capture CIC and radiolabelled anti- IgG for detection. 16 ELISA modi cations are now more popular and are commercially available. Although they are based on similar principles, the two assays are not equivalent. 17 In lupus nephritis these assays consistently give higher results than in patients with inactive disease. 14,18 However, Valentijn et al. found that rises in C1qFP had a low sensitivity and speci city for disease exacerbation in lupus (36% and 29%, respectively), resulting in a positive predictive value of only 51%. 12 Abrass et al. claimed a positive predictive value of 82% for disease exacerbation in SLE when the C1qFP went from negative to positive. 19 Coppo et al. reported that only 50% of their patients showed an increase in C1qFP prior to clinical exacerbation. 14 In contrast, they found that, in lupus nephritis, a rapid drop in C1qSP on treatment was a good prognostic feature and that this preceded recovery in serum complement levels. Several substances may interfere with these assays. C1q may bind polyanions (e.g. DNA, lipopolysaccharide),which can affect uid-phase assays. 20 C1qSP assays avoid these problems but RF can interfere instead. More recently, possible interference from anti-c1q autoantibodies 21,22 has been described. Detection of previously bound IC-associated complement Conglutinin-binding assays Conglutinin, a 750-kDa protein found in some ruminants, including cattle, binds to the high mannose group of the inactivated complement component C3bi. As such it is a C-type lectin. Conglutinin-binding assays utilize this calciumdependent C3bi-binding property of conglutinin to isolate ICs. Such assays were rst described in 1977 as solid-phaseassays. 23,24 Conglutinin is used as the capture phase in an ELISA. The complex is detected by means of anti-immunoglobulin. Like the C1q-binding assay, this assay is prone to interference, in particular from anti-collagen II antibodies. 25 As these antibodies are frequently found in connective tissue diseases, 26 the utility of the assay is restricted. One study has advocated the use of the ratio of calciumdependent to calcium-independent binding to overcome the interference from conglutininbinding antibodies. 27 Although popular in other countries, this assay has not been widely used in the UK. The Raji cell assay The Raji cell (derived from a B-lymphoblastic Burkitt lymphoma) assay uses the CR2 complement receptors found on this cell line to detect C3bi bound to ICs. Although one of the better assays, it has never been widely available in the UK. The major disadvantage is the need to use living cells. Anti-lymphocyte antibodies may interfere with the assay, 28 as may anti-dna. 29 A commercial alternative (the Raji Cell Replacement Assay) is available, which uses monoclonal antibody to C3 conversion products as the capture phase. This assay has been used to study nephritis in SLE. 30 CIC levels were found to be four times higher in proliferative glomerulonephritis than in membranous glomerulonephritis. Antiglobulin assays These assays rely on the property of RF, usually monoclonal, to interact strongly with aggregated IgG but only weakly with monomeric IgG. These assays differ from many others in being able to detect non-complement-binding ICs. Originally described as a radioimmune inhibition assay, 31 solid-phase assays have also been described. 32 Although functionally one of the better methods, the antiglobulin method has not

7 Immune complexes in routine clinical practice 259 found widespread application. It has been used in the investigation of autoimmune disorders. 31,33 High levels of monomeric IgG and intrinsic RF may interfere. INDIRECT ASSAYS: COMPLEMENT CONSUMPTION OR ACTIVATION These assays rely on various methods for the assessment of complement activation. C3/C4 Monitoring C3 and C4 levels in SLE has been used as an indicator of disease activity for many years. For example, in 1977, Davis et al. 34 noted that a fall in C4 levels tended to precede a fall in C3 levels. The authors found that, in some cases, complement levels were a more sensitive indicator of clinical change than the titre of antidouble-stranded DNA antibody. This approach is not applicable to all cases of SLE. Some SLE patients have a pre-existing C4 de ciency or, more commonly, a partial C4 de ciency; thus, C4 may be permanently low, even in periods of remission. Miescher et al. showed low C4 to be a consistent nding in HCV-associated cryoglobulinaemia (15/15 patients). 2 Hypocomplementaemia is a frequent nding in cryoglobulinaemia. This may in part be due to chronic activation in vivo, but is aggravated by IC-mediated activation in vitro. 35 Figure 3 also illustrates the use of C4 to monitor a patient with type II cryoglobulinaemia. Note that C4 is faster and easier to measure than cryoglobulinand that its level falls prior to the rise in cryoglobulin in the relapse episode. Measurement of C3 and C4 may thus be a mirror for the activity of ICs. It should be remembered, however, that there are other in uences on C3 and C4 levels, not least the involvement of complement in acute-phase response. C3, C4 and other complement components may be elevated as part of the acute-phase response. There may therefore be forces working in opposite directions to in uence the overall levels detectable in the serum. In vitro complement consumption assay This assay, also called anti-complementary activity, measures the ability of ICs to bind and activate complement, thus reducing the amount of functional complement remaining available for erythrocyte (sheep) lysis in haemolytic assays. 36 Interfering substances include C- reactive protein, heparin and DNA, alternative pathway activators (e.g. endotoxin) and certain enzymes (e.g. plasmin). 37 Furthermore, in order for the assay to operate, it is necessary to heatinactivate intrinsic complement completely in the test samples at 56 Ê C. This may lead to aggregation of immunoglobulinand false positive results. Interestingly, in a patient with C2 de ciency and SLE, this assay showed good correlation with other immunological measures of IC activity (complement levels, CIC by a capture ELISA, C3d), but could not be directly correlated with clinical activity. 38 In the study of Davis et al., ICs were associated with high DNA binding and active disease, but many patients with clinically active disease had no detectable ICs. 34 Total haemolytic complement (CH 50 ) CH 50 is a functional complement test based on the ability of the patient s complement to lyse IgG-coated sheep erythrocytes. Titration allows calculation of the amount of serum needed to achieve 50% lysis by spectrophotometry. It is envisaged that, in the presence of ICs, complement will be consumed in vivo. Thus, a low CH 50 would indicate the presence, and a high or normal CH 50 the absence, of ICs. Arguably, the use of CH 50 as a measure of ICs is ill-advised. The need for fresh samples is even more important here than in other IC assays and artefacts of sample decay are common. Furthermore, de ciency of any of the complement components will result in a low CH 50, such as the C4 de ciency frequently seen in SLE. Despite this, some favour its use as part of a panel for monitoring SLE. It is lowest in patients with renal involvement. 39 ANTIGEN-SPECIFIC ASSAYS Several studies have sought to identify the antigens involved in ICs in certain diseases, but not all of these have been successful. For example in 1977, Izui et al. found that, although 52% of SLE patients had CIC, only 6% had DNA/anti-DNA complexes. 40 The wide diversity of antigens potentially involved in IC disease makes it unlikely that this approach will ever be widely used; thus the approach is limited to research only. THE PROBLEM OF DISEASE NON-SPECIFICITY Several groups have shown that no single assay shows good discrimination across different IC diseases. The World Health Organization

8 260 Lock and Unsworth TABLE 3. Detection of immune complexes in disease expressed as a percentage of patients with a positive test result Disease Method SLE Vasculitis RA Leprosy Lymphoma C1q binding C1q solid phase Raji cell Conglutinin From Lambert et al. 33 Published with permission. RA=rheumatoid arthritis; SLE=systemic lupus erythematosus. (WHO) study (1978) 33 exempli es the problem (see Table 3). STANDARDIZATION AND QUALITY CONTROL Standardization of IC assays is a major problem. The most frequently used standard is heataggregated IgG, which has the advantage of being relatively simple to make. However, it does not correspond to a physiological complex. In 1984, a standard IC composed of tetanus and anti-tetanus in de ned proportions was proposed. 41 This promising approach does not appear to have been taken forward. There is no UK National External Quality Assurance Scheme for ICs. FINAL COMMENT The early enthusiasm for the detection of CIC has to a large extent waned, primarily because the use of non-antigen-speci c tests yields very little of diagnostic value. The view expressed by the WHO in ± `The detection of immune complexes is not essential in any clinical condition ± remains unchallenged in REFERENCES 1 Brouet JC, Clauvel JP, Danon F, Klein M, Seligmann M. Biologic and clinical signi cance of cryoglobulins. Am J Med 1974; 57: 775±88 2 Miescher PA, Huang Y-P, Izui S. Type II cryoglobulinaemia. Semin Hematol 1995; 32: 80±5 3 Agnello V, Chung RT, Kaplan LM. A role for hepatitis C virus infection in type II cryoglobulinaemia. N Engl J Med 1992; 327: 1490±5 4 Hartmann H, Schott P, Polzien F, Mihm S, Uy A, Kaboth U, et al. Cryoglobulinemia in chronic hepatitis C virus infection: prevalence, clinical manifestations, response to interferon treatment and analysis of cryoprecipitates. Z Gastroenterol 1995; 33: 643±50 5 Lunel F, Musset L, Cacoub P, Frangeul L, Cresta P, Perrin M, et al. Cryoglobulinaemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology 1994; 106: 1291±300 6 Pozzato G, Mazzaro C, Crovatto M, Modolo ML, Ceselli S, Mazzi G, et al. Low-grade malignant lymphoma, hepatitis C infection, and mixed cryoglobulinaemia. Blood 1994; 84: 3047±53 7 Ferri C, Marzo E, Longombardo G, Lombardini F, La Civita L, Vanacore R, et al. Interferon-a in mixed cryoglobulinemia patients: a randomised, crossover-controlled trial. Blood 1993; 81: 1132±6 8 Johnson RJ, Gretch DR, Couser WG, Alpers CE, Wilson J, Chung M, et al. Hepatitis C virusassociated glomerulonephritis. Effect of alphainterferon therapy. Kidney Int 1994; 46: 1700±4 9 Misiani R, Bellavita P, Fenili D, Vicari O, Marchesi D, Sironi PL, et al. Interferon alpha-2a therapy in cryoglobulinaemia associated with hepatitis C virus. N Engl J Med 1994; 330: 751±6 10 Levo Y, Gerovic PD, Kassab HG, Zucker- Franklin D, Franklin EC. Association between hepatitis B and essential mixed cryoglobulinemia. N Engl J Med 1977; 296: 1501±4 11 Bulphoni A. Mixed cryoglobulinemia and the hepatitis C virus [Editorial]. Minerva Med 1995; 86: 193±8 [in Italian; comment from Abstract] 12 Valentijn RM, Van Overhagen H, Hazevoet HM, Hermans J, Cats A, Daha MR, et al. The value of complement and IC determinations in monitoring disease activity in patients with systemic lupus erythematosus. Arthritis Rheum 1985; 28: 649±58 13 Coppo R, Bosticardo GM, Basolo B, Messina M, Mazzucco G, Stratta P, et al. Clinical signi cance of the detection of circulating ICes in lupus nephritis. Nephron 1982; 32: 320±8 14 Soltis RD, Hatz DE. The effect of serum immunoglobulin concentration on immune complex detection by polyethylene glycol. J Immunol Meth 1983; 57: 275±82 15 Zubler RH, Lange G, Lambert PH, Miescher PA. Detection of immune complexes in unheated sera by a modi ed 125 I-C1q binding test. Effect of heating on the binding of C1q by immune complexes and application of the test to systemic lupus erythematosus. J Immunol 1976; 116: 232±5 16 Hay FC, Ninehma LJ, Roitt IM. Routine assay for the detection of immune complexes of known

9 Immune complexes in routine clinical practice 261 immunoglobulin class using solid-phase C1q. Clin Exp Immunol 1976; 24: 396± Agnello V, Mitamura T. Detection of immune complexes in systemic lupus erythematosus with the C1q solid phase assay: correlation with ndna antibodies and hypocomplementemia. Clin Immunol Immunopathol 1987; 42: 338±43 18 Davis CA, Marder H, West CD. Circulating immune complexes in membranoproliferative glomerulonephritis. Kidney Int 1981; 20: 728±32 19 Abrass CK, Nies KM, Louie JS, Border WA, Glassock RJ. Correlation and predictive accuracy of circulating immune complexes with disease activity in patients with systemic lupus erythematosus. Arthritis Rheum 1980; 23: 273±82 20 Cooper NR, Morrison DC. Binding and activation of the rst component of human complement by the lipid A region of lipopolysaccharides. J Immunol 1978; 120: 1862±8 21 Antes U, Heinz H-P, Loos M. Evidence for the presence of autoantibodies to the collagen-like portion of C1q in systemic lupus erythematosus. Arthritis Rheum 1988; 31: 457±64 22 Uwatoko S, Mannik M. Low-molecular weight C1q binding immunoglobulin G in patients with systemic lupus erythematosus consists of autoantibodies to the collagen like region of C1q. J Clin Invest 1988; 82: 816±24 23 Casali P, Bossas A, Carpentier NA, Lambert PH. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by bovine conglutinin: conglutinin-binding test. Clin Exp Immunol 1977; 29: 342±54 24 Eisenberg RA, Theophilopoulos AN, Dixon FJ. Use of bovine conglutinin for the assay of immune complexes. J Immunol 1977; 18: 1428±34 25 Holmskov U, Haas H, Teisner B, Andersen O, Jensenius JC. Calcium-dependent and calciumindependent signals in the conglutinin-binding assay (KgBa) for immune complexes. In uence of anti-collagen antibodies. J Immunol Meth 1992; 148: 225±32 26 Morgan K. What do anti-collagen antibodies mean? Ann Rheum Dis 1990; 49: 62±5 27 Svendsen A, Holmskov U, Petersen PH, Jensenius JC. Difference and ratio plots: simple tools for improved presentation and interpretation of scienti c data. Unexpected possibilities for the use of the conglutinin binding assay in in ammatory rheumatic diseases. J Immunol Meth 1995; 178: 211±8 28 Osterland CK. Immune complexes in systemic lupus erythematosus. In: Espinoza LR, Osterland CK, eds. Circulating Immune Complexes: Their Clinical Signi cance. New York: Futura Publishing Co., 1983: 63±85 29 Tron F, Jacob L, Bach J-F. Binding of a murine monoclonal anti-dna antibody to Raji cells. Implications for the interpretation of the Raji cell assay for immune complexes. Eur J Immunol 1984; 14: 283±6 30 Robinson MF, Roberts JL, Jones JV, Lewis EJ. Circulating immune complex assays in patients with lupus and membranous glomerulonephritis. Clin Immunol Immunopathol 1979; 14: 348±60 31 Luthra HS, McDuf e FC, Hunder GG, Samoyoa EA. Immune complexes in sera and synovial uids with rheumatoid arthritis. Radioimmunoassay with monoclonal rheumatoid factor. J Clin Invest 1975; 56: 458±66 32 Davey MP, Korngold L. A solid phase assay for circulating immune complexes using monoclonal rheumatoid factors and peroxidase-linked protein A. J Immunol Meth 1981; 44: 87± Lambert PH, Dixon FJ, Zubler RH, Agnello V, Cambiaso C, Casali P, et al. A WHO collaborative study for the evaluation of eighteen methods for detecting immune complexes in serum. J Clin Lab Immunol 1978; 1: 1±15 34 Davis P, Cumming RH, Verrier-Jones J. Relationship between anti-dna antibodies, complement consumption and circulating immune complexes in systemic lupus erythematosus. Clin Exp Immunol 1977; 28: 226±32 35 Ueda K, Nakajima H, Nakagawa T, Shimizu A. The association of complement activation at a low temperature with hepatitis C virus infection in comparison with cryoglobulin. Clin Exp Immunol 1995; 101: 284±7 36 Haeney M. Tests for circulating immune complexes. In: Thompson RA, ed. Techniques in Clinical Immunology, 2nd edn. Oxford: Blackwell Scienti c, 1981: 170± World Health Organization. The role of immune complexes in disease. WHO Technical Report Series 606. Geneva: WHO, Erlendsson K, Traustado ttir K, Freysdo ttir J, Steinsson K, Jo nsdoâ ttir I, Valdimarsson H. Reciprocal changes in complement activity and immune complex levels during plasma infusion in a C2-de cient SLE patient. Lupus 1993; 2: 161±5 39 Lloyd W, Schur PH. Immune complexes, complement and anti-dna in exacerbations of systemic lupus erythematosus (SLE). Medicine 1981; 60: 208±17 40 Izui S, Lambert PH, Miescher PA. Failure to detect circulating DNA±anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus. Clin Exp Immunol 1977; 30: Nydegger UE, Svehag S-E. Improved standardization in the quantitative estimation of soluble immune complexes making use of an international reference preparation. Results of a collaborative multicentre study. Clin Exp Immunol 1984; 58: 502±9 42 IUIS/WHO Working Group. Laboratory investigations in clinical immunology: methods, pitfalls and clinical indications. Clin Exp Immunol 1988; 74: 494±503 Accepted for publication 1 September 1999