Laboratory testing for systemic autoimmune diseases

Anti-cellular antibodies of the antinuclear antibodies (ANA) family

Antinuclear antibodies (ANA), since first described around 60 years ago, are often analyzed, as prototypic autoantibodies, as part of the diagnostic evaluation of systemic autoimmune diseases, where there is a special association with connective tissue diseases. Connective tissue diseases include systemic lupus erythematosus (SLE), systemic sclerosis, Sjogren’s syndrome, poly-/dermatomyositis and mixed connective tissue disease (Sharp’s syndrome).

However, ANA occur with varying frequency in the various connective tissue diseases, and can also be detected in other diseases (e.g. juvenile idiopathic arthritis, autoimmune hepatitis). What is more, ANA can often be detected years before a connective tissue disease manifests, as well as in healthy people. ANA may also be induced by drugs or occur in connection with infections. The positive predictive value for a manifest systemic autoimmune disease is, therefore, extremely low for a randomly verified ANA titer (<5%), which is why the ANA diagnosis should only be carried out selectively if there is an appropriate suspicion. The gold standard for the detection of ANA is the indirect immunofluorescence assay (IFA) using the HEp-2 cell line and its variants, which is used for screening. This method, however, does not only detect autoantibodies directed against the cell nucleus, but also autoantibodies that recognize other cellular structures. This is why it is being attempted to replace the term “ANA” with a new designation, “anti-cellular antibodies of the ANA family” [6].

The IFA allows for different patterns to be differentiated that point to the recognized autoantigen. But the exact differentiation of the ANA specificity is realized by means of a two-stage detection method that uses immunoassays as a follow-up to a positive ANA IFA. The incidence of the most important specific autoantibodies and their association with connective tissue diseases and/or clinical pictures are shown in Table 1. Given the complexity of possible antibody combinations of different clinical pictures as well as the often low sensitivity and widely varying specificity of individual autoantibodies, a diagnosis becomes possible only after having looked at the overall picture of all laboratory results in combination with the patient’s history and clinical findings [1].

International recommendations regarding ANA diagnostics

Many institutions now use the new immunoassays for ANA screening, rather than the conventional IFA, because a larger number of patient samples can be processed faster and more easily this way. But the immunoassays available up to now have a very limited autoantigen spectrum (generally, up to ten autoantigens), while the HEp-2 cell contains a significantly larger number of nuclear and cellular autoantigens (approx. 100–150 autoantigens). It is against this background that the American College of Rheumatology (ACR) recommends in a current position paper the IFA in combination with the HEp-2 cell line as the continued gold standard of ANA screening [7]. Since the various ANA detection methods differ considerably in terms of methodology, autoantibody profiles detected, as well as sensitivity and specificity, there is uncertainty regarding standardization and the interpretation of incongruent test results [1, 8].

An international group of experts has, therefore, worked out recommendations on ANA screening and the interpretation of test results, proposing a two-stage process: the IFA is classified as a reference method for ANA screening, which is to be supplemented by specific immunoassays to differentiate autoantibody specificity in the case of positive results (1:160). The report on the results should include the detection method, the highest positive titer, as well as the nuclear and, if applicable, cytoplasmic pattern according to standardized terminology. If SLE is suspected, or if anti-dsDNA ab are present, the Farr assay or Crithidia luciliae IFA should be added to the array, because these methods have a maximum of specificity. The recommendations also particularly take into account the problem of incongruent test results, which is why the use of another established detection method should be considered, while ensuring the close exchange between clinicians and laboratory physicians [6, 9].

Automated ANA-IFA analysis systems

The IFA, as a reference method for ANA screening, comes with numerous disadvantages: the method is time-consuming, offers little in the way of automation and standardization, and requires an assessment by an experienced expert, who issues a final report based on his/her own subjective evaluation. It is, thus, particularly susceptible to errors. Keeping this in mind, various manufacturers have developed automated ANA-IFA analysis systems that can reduce the workload by up to 24% [10]. The systems already stand out for being able to discriminate well between positive and negative ANA results, a fact borne out by achieving a sensitivity of 96.7% and a specificity of 89.2%. The signal intensity also correlates well titers analyzed with conventional methods (Spearman’s rho: 0.672–0.839; p<0.0001 for all systems). Some systems are already capable of automated pattern recognition, but the level of correlation with manual evaluation is still borderline (52%–79%) [11].

ACR/EULAR 2013 classification criteria for systemic sclerosis

In up to 50% of patients with manifest systemic autoimmune diseases, no definitive diagnosis is possible in the first 12 months. Accordingly, one often talks of undifferentiated diseases in such cases, such as undifferentiated arthritis or undifferentiated connective tissue disease [1]. Against this background and in light of significantly enhanced therapeutic options, recent years have seen special efforts to develop new criteria for the diagnosis and/or classification of systemic autoimmune diseases that would allow for earlier diagnosis and classification. Autoantibodies are given special consideration in this context: being characteristic markers of autoimmunity, they are relevant not only in diagnostic and prognostic terms, but also, in part, with respect to pathophysiology. Given the inadequacies of existing classification criteria, new classification criteria have now been developed, and published (see Table 2), for SSc as part of an EULAR/ACR initiative.

Apart from skin involvement and clinical findings, these new classification criteria now also account for autoantibody results and capillary microscopy. The following SSc-associated autoantibodies have been included in the new classification system: anti-centromere ab, anti-topoisomerase I [anti-Scl-70] ab and anti-RNA polymerase III ab, which should be determined in case of clinical suspicion [12].

Current Chapel Hill consensus nomenclature of vasculitis

Based on the results of a new consensus conference in 2011, the Chapel Hill nomenclature of vasculitis has been completely revised and published (also see Figure 1) [13]. Following the trend of doing away with eponyms when naming diseases, the new Chapel Hill nomenclature, too, replaces familiar disease designations with new names. Thus, Wegner’s granulomatosis is now called “granulomatosis with polyangiitis” (GPA). This practice of naming diseases on the basis of the pathological results, for example, has also changed the name of the Churg-Strauss syndrome to “eosinophilic granulomatosis with polyangiitis (EGPA)”. For other entities, such as Takayasu’s arteritis and Kawasaki disease, however, the known eponyms have been kept. The vasculitis classification now includes seven different categories. New elements of this classification include, for example, the demarcation of vasculitis of individual organs (e.g. primary cerebral vasculitis or isolated vasculitis) into a common group, or the differentiation of separate categories for secondary vasculitis in connection with systemic diseases (e.g. rheumatoid vasculitis) as well as those of other known etiology (e.g. those induced by infection or drugs). One important change concerns small-vessel vasculitis. Under the Chapel Hill classification, microscopic polyangiitis, GPA and EGPA are now grouped under the heading of “ANCA-associated vasculitides (AAV)” and thus differentiated from immune-complex vasculitides. Now, anti-GBM disease (formerly: Goodpasture syndrome) and IgA vasculitis, which used to be known as Henoch-Schönlein purpura, are included with immune-complex vasculitides of small vessels. The disease definitions under the Chapel Hill nomenclature are primarily derived from the underlying pathology (vessel size, composition of the inflammatory infiltrate, etc.) and pathogenesis (e.g. antibody-associated, immune-complex-mediated, infection-related, etc.), and only partially include characteristic clinical features. Thus, the Chapel Hill nomenclature does not replace classification criteria, such as the still-valid ACR classification criteria for vasculitides.

Figure 1:

Distribution pattern of vasculitides according to the new nomenclature. Modified according to [13].

Antineutrophil cytoplasmic antibodies (ANCA)

Similarly to ANA, the recommendation for ANCA diagnostics also involves the combination of IFA (screening) with immunoassays to detect specific antibodies against proteinase-3 (PR3) and myeloperoxidase (MPO). There is a distinction between c-ANCA (cytoplasmic pattern), p-ANCA (perinuclear pattern) and x-ANCA (atypical pattern), where the binding behavior of antibodies is examined on ethanol- or formalin-fixed granulocytes. Here, over 90% of anti-PR3 ab in the IFA exhibit a c-ANCA pattern, and only rarely a p-ANCA pattern, while anti-MPO ab mostly have a p-ANCA pattern (approx. 90%), and only rarely a c-ANCA pattern. Every positive or unclear IFA outcome should always and absolutely be clarified further through specific immunoassays that are available – or both procedures should be run in parallel from the start. X-ANCA do not react against PR3 or MPO, and are not associated with vasculitis. They are frequently found in patients who suffer from other autoimmune diseases, such as ulcerative colitis, primary sclerosing cholangitis or autoimmune hepatitis, and rarely in patients with Crohn’s disease, RA or connective tissue diseases [14, 15].

If an ANCA-associated vasculitis (AAV) and, in particular, a pulmonary-renal syndrome are suspected, it is of special diagnostic importance to test for ANCA and anti-glomerular basement membrane antibodies (anti-GBM ab) quickly. Often, however, these autoantibodies are only analyzed as part of routine diagnostics and are not available as emergency parameters. This can cause a significant delay in arriving at a diagnosis particularly for critical patients. One study, therefore, involving 260 consecutive patients suspected of AAV, looked at the accuracy of rapid tests (dot blot method, with results within two hours) when it comes to analyzing anti-PR3 ab, anti-MPO ab and anti-GBM ab by means of standard diagnostic procedures (IFA and ELISA) [16]. For a total of 74 patients in this study, the diagnosis was AAV (n=62) or anti-GBM disease (n=12). The dot blot method and the ELISA both identified all twelve cases of anti-GMB disease reliably, with two false-positive results. The rapid dot blot test was positive for ANCA in 56 of 62 AAV patients (sensitivity 90%, NPV 97%), with five false-positive results (specificity 97%, PPV 90%). The Phadia ELiA test for anti-PR3s or anti-MPOs produced positive results for 57 of 62 AAV patients (sensitivity 92%, NPV 97%), with five false-positive results (specificity 97%, PPV 88%). The routine ELISA yielded similar results (sensitivity 94%, specificity 97%, PPV 88%, NPV 98%) [16].

Thus, the findings from the rapid dot blot test for anti-GBM ab, anti-PR3 ab and anti-MPO ab and those obtained through established routine methods were in perfect agreement. Where such diagnoses are suspected, the use of these rapid tests, particularly in intensive-care units, may produce a diagnosis for critical vasculitis patients sooner.

Anti-ferritin antibodies in connection with polymyalgia rheumatica and giant cell arteritis

The diagnosis of polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) is often difficult due to the lack of biomarkers. A working group, therefore, looked for new autoantibodies in these patients using protein arrays [17]. It was thanks to this method that autoantibodies against 27 N-terminal amino acids (19–45) of the heavy chain of the human ferritin protein were detected for the first time in PMR and GCA patients. To analyze the significance of these autoantibodies as biomarkers, the sera of PMR and GCA patients were tested for the presence of anti-ferritin ab by means of ELISA. Positive anti-ferritin ab results were found in 92% of untreated PMR and GCA patients, and in 69% of PMR and GCA patients with acute episodes. By contrast, anti-ferritin ab were identified only in 29% of SLE, 3% of RA, 6.5% of NHL and 1% of healthy blood donors who served as controls [17]. These very good results, however, have meanwhile been put into perspective by another study, where anti-ferritin ab were found in only 72% of GCA patients with a histologically confirmed diagnosis. Furthermore, anti-ferritin ab were detected in only 41.3% of GCA patients with a negative histology, which was in the range of what had been found in control patients with other diagnoses (31.9%). Having said this, this study, too, found anti-ferritin ab in only 2.5% of healthy test subjects [18]. The significance of these autoantibodies with respect to the heavy chain of the human ferritin protein, thus, remains unclear and will have to be studied further.

Anti-carbamylated protein (anti-CarP) antibodies as new biomarkers

In addition to the rheumatoid factor (RF), the current 2010 ACR/EULAR classification criteria for rheumatoid arthritis (RA) also take into account anti-citrullinated protein/peptide antibodies (ACPA) as a criterion [19]. Both RF and ACPA can be detected many years before RA manifests itself, but around 20% of RA patients do not develop any of these autoantibodies (“seronegative” RA). In light of this, further biomarkers are needed that allow for an early and reliable diagnosis and that, ideally, are also pathophysiologically relevant. Such new biomarkers might be autoantibodies against carbamylated proteins (anti-CarP), which can be detected several years before RA manifests in the serum of some RF and ACPA-negative RA patients [20]. Carbamylation is a post-translational modification that converts lysine to homocitrulline. In this study, sequential serum samples from 79 RA patients were analyzed who had regularly donated blood before the manifestation of RA. By using two different antigen systems, anti-CarP antibodies were detected in about one third of these RA patients. Five of the 79 RA patients examined tested positive for anti-CarP antibodies exclusively, but had negative findings for ACPA and RF (Figure 2).

Figure 2:

Distribution pattern of ACPA, IgM-RF and anti-CarP antibodies in 79 RA patients. Modified according to [20].

Anti-drug antibodies (ADAb)

The therapeutic options for RA have grown substantially thanks to biologics. TNF inhibitors, in particular, have proved effective in the treatment of a variety of chronic inflammatory diseases. However, more than a third of patients do not respond to an anti-TNF therapy (primary treatment failure), or develop a secondary treatment failure following an initial response. A good therapeutic effect has been observed in some of these patients after switching to a different TNF inhibitor, which is why the presence of anti-drug antibodies (ADAb) is believed to be associated with a loss of efficacy. ADAb against biologics represent the immune system’s response to the administration of exogenous proteins, and are directed against the structures of the respective biologic. ADAb were observed in 28% of RA patients following treatment with adalimumab over a period of 3 years [21]. In the meantime, the biological significance of ADAb has been worked out in numerous studies with respect to the correlation of ADAb levels, reduced drug levels and loss of efficacy [22]. In particular, it has been demonstrated formally how ADAb neutralize the functional part of the TNF inhibitor adalimumab, which ends up inhibiting the biological activity of the anti-TNF antibody [23]. Since there are now various test systems for detecting ADAb and TNF inhibitor levels in the serum, and in light of these findings, it has been proposed that patients be monitored for drug levels and ADAb (see Figure 3) [22].

Figure 3:

Proposal for a decision algorithm taking into account the TNF inhibitor concentration and ADAb test. Modified according to [22].

HLA-B27

So far, only the HLA-B27 test has been established as a biomarker for spondyloarthritides – it has been included in the current classification criteria. But HLA-B27 can also be detected in around 10% of healthy individuals, which limits the specificity and, thus, the clinical relevance. Several methods for detecting HLA-B27 have been established: flow cytometry using monoclonal antibodies, PCR methods using sequence-specific primers (SSP), and the sequencing of HLA-B27 gene as the gold standard. Flow cytometry represents a fast and cost-efficient method. However, the cross-reactivity of the antibodies used with other surface antigens, such as HLA-B7 and HLA-B37, limits the specificity of the process [24]. For FD705 antibody assays, it has been shown that an optimization of the cutoff of the mean fluorescence intensity (MFI) can achieve a sensitivity of 98.2% and a specificity of 97.6% – considered sufficient for routine clinical purposes. Another frequently used antibody is the GS145.2 antibody assay: With the manufacturer’s cutoff, it achieves a sensitivity of 100%, but its specificity is low at 71.4%. But a modified cutoff, as proposed in the study, can help achieve a significantly improved specificity of 88.6%, while the sensitivity is decreased only slightly to 95.2%. Nevertheless, when using the GS145.2 antibody assay, a follow-up test should be conducted with an FD705 antibody assay in a specific critical range of the MFI. This can produce a sensitivity of 98.8% and a specificity of 97.6% [25].

The standard PCR with sequence-specific primers (SSP) and the more practical real-time PCR methods based on it represent a further, highly specific and highly sensitive alternative. Compared to the gold standard of DNA sequencing, a sensitivity of 99.6% and a specificity of 100% is reached, depending on the primer mix employed [25]. But the choice of primer can also create limitations, because not all subtypes are captured all the time: When using the PCR protocol according to Olerup, the subtypes B*27:12, B*27:16, B*27:18 and B*27:23 are not amplified, and using the PCR according to Dominguez, this is true of the subtypes B*27:07, B*27:14, B*27:19 and B*27:21 [26]. Since alleles occur with varying frequency in different ethnic groups, the sensitivity of one and the same test can vary in patients of different ethnic backgrounds, yielding an increase in false-negative results [26]. The EUROArray HLA-B27-Direct test from the company Euroimmun represents a further possible improvement. According to the manufacturer, this test captures all known HLA-B27 alleles. It also detects the non-disease-associated subtypes HLA*B27:06 and HLA*B27:09 [27].

Anti-CD74 antibodies

Given the prevalence of HLA-B27 in 10% of the healthy population, a working group looked for new autoantibodies and/or autoantigens in patients with ankylosing spondylitis (SpA) using protein arrays [28]. It was through this method that autoantibodies against CD74 protein (anti-CLIP ab) were first detected in these patients. After establishing a specific ELISA, the frequency of anti-CD74 antibodies was analyzed in different patient cohorts. Anti-CD74 IgG autoantibodies were found in SpA patients with a frequency of 69%. These autoantibodies were also detected in 45% of psoriatic arthritis patients without axial disease, 11% of RA patients, 15% of SLE patients, 2.5% of HIV patients and 0.8% of healthy blood donors. These promising results were also confirmed by another working group who worked with different patient cohorts and managed to confirm anti-CLIP ab in 85.1% of patients with axial spondyloarthritis, while only 7.8% of the control patients exhibited these autoantibodies. No HLA-B27 was detected in 23.6% of patients with axial spondyloarthritis, while only 14.9% of these patients did not show any signs of anti-CLIP ab at all. Based on these findings, a sensitivity of 85.1% and a specificity of 92.2% (LR+: 10.8; LR–: 0.08) were calculated for the positive anti-CLIP ab detection with respect to a diagnosis of axial spondyloarthritis [29]. Thus, autoantibodies against CD74 (anti-CLIP ab) are new biomarkers that are much more promising in terms of spondyloarthritides with axial disease. However, the significance of this will have to be evaluated in further studies.

Anti-MCV antibodies

Autoantibodies against mutated citrullinated vimentin (anti-MCV ab) are part of the group of anti-citrullinated protein/peptide antibodies (ACPA), and – with a specificity of 94% – represent a key marker for confirming a diagnosis of rheumatoid arthritis [30]. But anti-MCV ab have also been observed in SpA patients. A 2012 study of a total of 43 SpA patients found significantly elevated serum levels of anti-MCV ab compared to the healthy control group [17.3 U/mL versus 8.9 U/mL (p<0.01)]. Where the recommended cutoff of the manufacturer (>20 U/mL) was applied, 37% of SpA patients exhibited an elevated anti-MCV ab level, while no one in the healthy control group exceeded the cutoff [31]. These data give rise to the assumption that the detection of anti-MCV ab could reach a high specificity regarding a diagnosis of SpA in connection with a positive anti-MCV ab finding and typical clinical symptoms, but without any indication of RA. However, the small study size is a considerable limiting factor, which means that the relevance of this observation, including precise analyses of sensitivity and specificity, will have to be studied further.

Anti-β2-glycoprotein I antibodies

The current and revised classification criteria for the antiphospholipid syndrome (APS) dating from 2006 include, with respect to autoimmunity diagnostics, testing for anti-cardiolipin ab (aCL) or anti-β2-glycoprotein I antibodies (anti-β2GPI), as well as the lupus anticoagulant, with a positive test result expected to be confirmed after twelve weeks at the earliest [32]. So far, only the isotypes IgG and IgM have been considered in connection with the aCL and anti-β2GPI ab, even though an association with clinical manifestations of APS and a special pathophysiological relevance in animal models have been known in the context of the IgA isotype of these antibodies. Therefore, a working group studied the frequency of isolated anti-β2GPI-IgA antibodies on three large patient cohorts (a total of 5871 patients) [33]. Overall, a positive anti-β2GPI-IgA titer was identified in 198 patients of whom 57 patients tested positive only for the anti-β2GPI-IgA antibodies. At least a clinical APS manifestation was detected in 70.1% of these patients. There was also a positive correlation with the occurrence of venous and arterial thrombosis. What is more, in vivo experiments on mice revealed a greater thrombus formation after the transfer of anti-β2GPI-IgA antibodies.

It must, therefore, be noted that an analysis of the anti-β2GPI-IgA antibodies is recommended in the case of clinical suspicion and in the absence of proof of the lupus anticoagulant and the aCL and/or anti-β2GPI antibodies of the isotypes IgG and IgM.

Diagnostic algorithm

The new concept of IgG4-related diseases (IgG4-RD) has been developed over the past 10 years. These are now grouped into a separate disease entity [34]. IgG4-RD are distinguished by a special pathohistological pattern (tissue infiltration with IgG4-expressing plasma cells, storiform fibrosis, obliterative phlebitis, as well as well as mild to moderate eosinophilia) [35]. Multisystem fibroinflammatory diseases are common in patients affected (simultaneously or consecutively), impacting multiple organ systems and body regions.

The etiology and exact pathogenesis of these diseases are still unknown. Aside from an autoimmune etiology, an allergic cause has also been discussed, one that produces a dysregulation of T-cells, including a TH2 response and accompanied by consecutive tissue infiltration with IgG4-expressing plasma cells. The diagnosis is made after looking at the entire clinical picture, imaging, histology and laboratory results. Elevated IgG4 serum levels (>135 mg/dL) may be indicative of an IgG4-RD. But only around 80% of patients exhibit elevated IgG4 levels in the serum, and elevated IgG4 levels are also found in approximately 5% of the general population.

The most important finding, therefore, is the histology test that shows an increase in IgG4-expressing plasma cells in the tissue, which requires special staining [34, 35]. International consensus recommendations for histology diagnostics were published in 2012. Together with the diagnostic criteria published in Japan in 2011, they make up the current diagnostic basis. Based on the key aspects of the two publications, a diagnostic algorithm has been proposed, which can be seen in Figure 4 [36].

Figure 4:

Diagnostic criteria for IgG4-RD. Modified according to [36].

Determination of IgG4 serum concentrations

A key criterion for diagnosing an IgG4-RD, it is believed, is an elevated IgG4 serum concentration >135 mg/dL. But this is observed only in around 80% of patients with a histologically confirmed IgG4-RD [34–36]. Furthermore, consecutive IgG4 tests on the same patient yielded widely differing results that did not correspond to the clinical findings – this was attributed to a so-called “high dose hook effect” (or “prozone effect”). Against this background, a working group analyzed the serum samples of an IgG4-RD patient cohort systematically with respect to the “prozone effect” in the IgG4 analysis [37]. It was found that the IgG4 concentrations in the initial analysis had been too low in 10 out of the 38 serum samples studied. Through a repeat analysis of serum dilutions, it was further found that all false-low IgG4 serum concentrations were due to a “prozone effect”, which could have been prevented by way of appropriate dilution stages.

Autoimmune disease episodes versus infection

In clinical practice, the distinction between an autoimmune disease episode and an infection is common, but also often very difficult due to similar clinical symptoms. Markers, therefore, with which these situations can be differentiated would be helpful so as to allow for a sound treatment decision (intensification of immunosuppression versus anti-infective treatment). It is in this context that the analysis of the procalcitonin (PCT) concentration has become routine diagnostic practice: A positive result indicates an underlying systemic, bacterial infection. The significance of PCT in such situations has meanwhile been studied in two meta-analyses [38, 39]. One of the meta-analysis, on the basis of nine studies on the presence of bacterial infection, determined a sensitivity of 0.75 (95% CI 0.63–0.84) and a specificity of 0.90 (95% CI 0.85–0.93) for the PCT test, as opposed to a sensitivity of 0.77 (95% CI 0.67–0.85) and a specificity of 0.56 (95% CI 0.25–0.83) for the CRP test. For this study, a positive likelihood ratio of 7.28 (95% CI 5.10–10.38) and a negative likelihood ratio of 0.28 (95% CI 0.18–0.40) were calculated for the PCT test [38]. Similar results were also found in the second meta-analysis with respect to the diagnostic value of the PCT test in connection with osteomyelitis and septic arthritis [39]. In summary, therefore, the result of a clearly elevated PCT level can make a valuable contribution in clinical practice to the differentiation between an autoimmune disease episode and bacterial infection. However, the other findings regarding the patient must still be taken into account in the differential-diagnostic classification. It bears mentioning that a PCT result that is within the norm does not rule out bacterial infection reliably under any circumstances.

Masking of the acute phase markers by immunosuppression

Immunosuppressive treatment can substantially impair the immune response to pathogens, which can cause the acute phase markers to be suppressed and the serological response to vaccinations to be diminished. One meta-analysis, for example, has shown for RA patients treated with rituximab that the response rate after a pneumococcal vaccination (measured by the increase in the antibody titer) is reduced for the serotype 6B [OR 0.25 (95% CI 0.11–058)] as well as for the serotype 23F [OR 0.21 (95% CI 0.04–1.05)] [40]. A detailed drug history is therefore essential for immunosuppressed patients suspected of an infection. In clinical practice, the extent of the inflammatory response is measured particularly by way of CRP and BSR. However, these parameters can be influenced substantially by conventional and biological immunosuppressants and steroids, which means that an increase that would be commensurate with the infection does not occur or is significantly lower. Since the release of CRP in the hepatocytes is regulated by IL-6, it is not surprising that the acute phase markers are suppressed, or masked, during IL-6-inhibiting treatment. Thus, various case reports contain accounts of a lack of increase in and/or suppression of CRP despite a systemic bacterial infection when the patient undergoes treatment with the anti-IL6-receptor antibody tocilizumab [41, 42]. Whether or not the PCT test is a reliable diagnostic marker in this situation is the subject of current studies.

Immunmonitoring using multiparameter flow cytometry

Opportunistic infections can manifest in patients undergoing immunosuppressive treatment due to excessive immunosuppression. So far, no comprehensive immunomonitoring has been established for such patients that would allow for therapy control and prevent excessive immunosuppression. Multiparameter flow cytometry allows for the simultaneous phenotypic characterization of many different cell populations in peripheral blood. This may possibly yield a more precise and individual manner of immunomonitoring in connection with longitudinal analysis. But this requires a standardization and validation of the method on the basis of larger patient populations. Given the special pathophysiological relevance and the effects of many immunosuppressants, typical surface markers have been proposed for the analysis of T- and B-cell subpopulations by means of multiparameter flow cytometry, which is to be investigated in future studies (also see Table 3) [43]. In a prospective, single-center study, 39 RA patients, who were given 1 g rituximab once every 15 days, were examined every 2 months until reinduction, and the B-cell subpopulations were analyzed. It was shown that it was possible to predict another episode within 4 months on the basis of the increase in CD19+/CD38++/CD24++ transitional B-cells (p=0.007) and in CD19+/CD27+ B-memory cells (p=0.01). Monitoring individual subpopulations of immune cells, thus, could become an effective tool of therapy control in the future [44].