Assessment Of Complement Activation Research Articles

Pitfalls in complement analysis: A systematic literature review of assessing complement activation

The complement system is an essential component of our innate defense and plays a vital role in the pathogenesis of many diseases. Assessment of complement activation is critical in monitoring both disease progression and response to therapy. Complement analysis requires accurate and standardized sampling and assay procedures, which has proven to be challenging. We performed a systematic analysis of the current methods used to assess complement components and reviewed whether the identified studies performed their complement measurements according to the recommended practice regarding pre-analytical sample handling and assay technique. Results are supplemented with own data regarding the assessment of key complement biomarkers to illustrate the importance of accurate sampling and measuring of complement components. A literature search using the Pubmed/MEDLINE database was performed focusing on studies measuring the key complement components C3, C5 and/or their split products and/or the soluble variant of the terminal C5b-9 complement complex (sTCC) in human blood samples that were published between February 2017 and February 2022. The identified studies were reviewed whether they had used the correct sample type and techniques for their analyses. A total of 92 out of 376 studies were selected for full-text analysis. Forty-five studies (49%) were identified as using the correct sample type and techniques for their complement analyses, while 25 studies (27%) did not use the correct sample type or technique. For 22 studies (24%), it was not specified which sample type was used. A substantial part of the reviewed studies did not use the appropriate sample type for assessing complement activation or did not mention which sample type was used. This deviation from the standardized procedure can lead to misinterpretation of complement biomarker levels and hampers proper comparison of complement measurements between studies. Therefore, this study underlines the necessity of general guidelines for accurate and standardized complement analysis.

Assessment of complement activation in the monkey: An in vitro model

Assessment of complement activation in the monkey: An in vitro model

Assessment of complement activation during membrane‐based plasmapheresis procedures

Previous studies have suggested that plasmapheresis procedures using a separation membrane may activate the complement system and release anaphylatoxins. This study determines the content in C3a/C3a(des Arg) and C5a/C5a(des Arg) in plasma donations obtained by the new Haemonetics Filter Core (FC) procedure and compares it to Baxter Autopheresis C (Auto-C). FC performs sequential blood centrifugation and plasma filtration on a microporous polyethersulfone membrane, while Auto-C removes blood cells by simultaneous gravitation and filtration on a rotating nylon membrane. One group of 34 donors donated on FC and two groups of 30 and 10 donors on Auto-C. Plasma aliquots were taken from the plasma units within 30 min of the end of the collection procedures, frozen at < -30 degrees C and assessed for C3a and C5a at various time points of storage. Mean C3a/C3a(des Arg) in FC plasma (N = 34) was 1,151 (range: 526-2,991), 1,092 (range: 349-3498), and 507 (range: 307-815) ng/ml at time of collection and after 6 and 12 months of storage, respectively. Respective C5a/C5a(des Arg) was 26.6 (range 4.9-74), 18.9 (9.5-42.6), and 30.9 (range: 10.7-62.3) ng/ml. Mean C3a/C3a(des Arg) was higher in Auto-C (P < 0.001): 4,724 ng/ml (N = 10; range: 2,400-7 ,360) and > 4,149 ng/ml (N = 30; 2,408- > 6,430) after 3 and 18 months storage, respectively. Mean C5a/C5a(des Arg) was 32.1 ng/ml (N = 30; range: 10.6-57.2) after 18 months of storage. Complement activation in FC plasmas appears limited compared to Auto-C, suggesting better biocompatibility of this collection device and/or a favourable impact of the sequential cell centrifugation/filtration technology used. Further studies are needed to explain differences in complement activation between apheresis procedures and to assess clinical impacts, if any.

Assessment of complement activation in autologous blood during cardiac surgery

Assessment of complement activation in autologous blood during cardiac surgery

Complement activation by polymethyl methacrylate minimized by end-point heparin attachment

After intraocular lens implantation, despite good clinical results, many cataract patients develop a chronic uveitis, caused by an inflammatory response to the implant. One way to improve the biocompatibility of the intraocular lens is to modify the surface by end-point heparin attachment. This study shows that complement activation caused by poly(methyl methacrylate) can be diminished by end-point heparin attachment, as demonstrated by a significant reduction in the generation of C3a and fluid phase terminal complement complexes. It suggests that assessment of complement activation is a good indicator of the biocompatibility of intraocular lenses.

Methods for assessing complement activation in the clinical immunology laboratory

Complement activation is a key component of the pathogenesis of immune-mediated tissue damage in many diseases. Assessment of complement activation in current practice is largely based on the measurement of intact C3 and C4 or the determination of complement haemolytic function. These parameters reflect activation only indirectly, are insensitive and open to influence by factors other than complement conversion. New approaches to evaluate complement activation directly using sensitive techniques have been developed, and several could be adopted easily in most laboratories. These concentrate on the detection of activation fragments, neoantigens or complexes that only arise as a direct result of complement activation. The wide application of these techniques in research and clinical practice would enhance our understanding of the pathogenesis of a range of inflammatory and infectious diseases.

Assessment of complement activation in clinical samples comparison of immunochemical and functional measurements of complement components with quantitation of activation fragments

We have compared functional and immunchemical measurements of complement components with assays measuring the generation of activation fragments, for the assessment of classical pathway activation in vitro and in vivo. The generation of the C3a, C3b/C3bi cleavage fragments of C3, and of the C4d cleavage fragment of C4 measured by ELISA and RIA, was correlated with the decrease in total complement hemolytic activity (CH50) and in functional activity of C3 and C4 in normal human serum in which the classical pathway had been activated with aggregated IgG. The decrease in CH50 in in vitro activated serum was also correlated with the generation of C5a and soluble SC5b-9 complexes. In contrast, no or little increase in the concentration of C3a, C3b/C3bi and C4d was observed in plasma samples from patients with low CH50 and with low levels of immunochemical C3 and C4, indicating that fragment quantitation assays provide no information on the presence and extent of complement activation in vivo. Analysis of samples from patients expressing the four C4 genes and patients having one or two C4 null alleles indicated that a ratio of hemolytic C4 to C2 ≥ 1 was indicative of complement activation without C4 deficiency, whereas a ratio below 1 was indicative of C4 deficiency with or without classical pathway consumption. Classical pathway activation and C4 deficiency in clinical samples are best predicted by the concommitant assessment of immunochemical levels of C3 and C4 and hemolytic levels of C4, C2 and C3.

Assessment of complement activation in vivo.

Hemolytic assays that measure the functional integrity of the complement system and the quantitation of individual components by immunochemical techniques have been widely used in the past for the assessment of in vivo complement activation. However, the complement system comprises a large number of interacting serum proteins which are subject to independent synthetic and catabolic processes. The fact that complement proteins are rapidly metabolized under in vivo conditions adds to the complexity of complement analysis. Assays that are based on monoclonal antibodies with specificities for activation-dependent neoepitopes now allow the direct determination of complement fragments in plasma. These methods are superior to the quantitation of native proteins. Several parameters that differentially affect the generation or the catabolism of individual complement activation products still have to be taken into account when elevated plasma levels of complement fragments suggest in vivo complement activation. These factors include the binding to complement fragment receptors, the degradation by serum proteases and renal or hepatic clearance. An accurate estimation of complement activation in vivo requires the simultaneous determination of both the native components and the activation products.

Activation of complement by hemodialysis membranes: Polyacrylonitrile binds more C3a than cuprophan

Conventionally, complement activation by hemodialysis membranes has been determined by measuring fluid phase C3a. Based on such measurements, polyacrylonitrile (PAN) membranes have been classified as weak activators compared to cuprophan. Previous studies have demonstrated, however, that PAN adsorb fluid phase C3a. Based on that observation, we hypothesized that complement activation by PAN might be artifactually underestimated if relatively large amounts of C3a remained membrane bound. In the present study, a method that allows the simultaneous quantification of both fluid phase and membrane bound C3a was used to assess complement activation by PAN and cuprophan. Pieces of membrane were incubated with C3-depleted serum that had been repleted with radiolabeled C3. Subsequently, the supernates and membranes were subjected to SDS-PAGE, and complement activation was quantified by determining the radioactivity of the C3a bands in the gel. The results showed that while the serum exposed to cuprophan membranes contained almost five times more C3a than that exposed to PAN, approximately 80 times more C3a was bound to the PAN membranes. Consequently, the total amount of C3a generated in the presence of PAN was higher than that generated in the presence of cuprophan. We conclude that assessment of complement activation by hemodialysis membranes using fluid phase C3a measurements alone may be misleading.

Assessment of complement activation in psoriasis by use of complement fragment C4d and Bb measurement

Assessment of complement activation in psoriasis by use of complement fragment C4d and Bb measurement

Review: assessment of complement activation in clinical immunology laboratories: time for reappraisal?

Review: assessment of complement activation in clinical immunology laboratories: time for reappraisal?

Assessment of complement activation during extracorporeal circulation by measurement of complement split C3d.

The present report summarizes our previous experience with the effect of various dialyzer membranes on leukocyte count, plasma C3d and C5a/C5ades-Arg, and total hemolytic complement. A relationship between the formation of C5a/C5ades-Arg within the dialyzer and hemodialysis leukopenia was demonstrated by comparing different membranes. Arterial plasma C3d was found to be a useful cumulative marker of complement activation during hemodialysis. C3d was shown to be generated in the artificial kidney with kinetics resembling the formation of C5a/C5ades-Arg. Total hemolytic complement transitorily decreased with all membranes, but did not reflect dialyzer biocompatibility. It is suggested that determination of complement-split C3d outdates previous more elaborate estimates of complement activation during hemodialysis.

Generation of Complement C3d within Artificial Kidneys

Complement activation during hemodialysis is related to dialysis discomfort and hypoxemia, and has been suggested to contribute to the occurrence of hypersensitivity-like symptoms, increased cardiovascular morbidity and chronic pulmonary disease. Assessment of complement activation by measurement of anaphylatoxin C5a is complicated by the short half-life of this compound. Arterial plasma C3d has previously been shown to be a suitable cumulative marker of complement activation during hemodialysis, and the present study was conducted to further elucidate this finding. Accordingly, arterial and dialyzer effluent plasma C3d was determined in 12 patients during a routine hemodialysis using saponified cellulose ester membrane. Hemodialysis was associated with transitory leukopenia and progressive increase in arterial plasma C3d. Generation of C3d within the dialyzer was demonstrated after 15, 30 and 60 min, and maximal generation rate coincided with the nadir of leukopenia at 15 min. The study documents the generation of C3d during hemodialysis, and adds further evidence for the formation of C3 convertase on dialyzer membranes.

Evaluation pratique de l'implication du complément au cours des réactions anaphylactoïdes

While no single test is entirely satisfactory for the investigation of anaphylactoid response to intravenous anaesthetic drugs, changes in plasma chemistry are particularly easy to measure and may be carried out at centres remote from the clinical incident. Analysis should be carried out on a sequence of blood samples taken into EDTA over the 24 hours following such reaction. Few reactions are classical Type I, IgE antibody mediated, reactions and most involved the complement proteins in some specific manner. By measuring changes in the levels and activity of the complement proteins, particularly C3, C4 and C5, between samples, it is possible to deduce the likely mechanism of a reaction and thus to advise more wisely on future treatment of the patient. Although levels of any plasma protein may be readily measured by single radial immunodiffusion (Mancini technique) using appropriate specific antisera, one assessment of complement activity (degree of conversion) has until now largely been restricted to technically exacting Laurell immunoelectrophoresis. Two simple immunoelectrophoretic techniques are described here which do not require such expertise.