Issue link: https://ahint.epubxp.com/i/574474
27 ASHI Quarterly Third Quarter 2015 S C I E N T I F I C C O M M U N I C A T I O N S The lectin and the alternative pathways are initiated mostly by microbial components as part of the innate immune system, which helps phagocytic cells to clear pathogens from the body The classical pathway can be initiated by specific antibodies recognizing foreign antigens including HLA determinants on donor cells resulting in AMR This pathway is the primary concern for the transplant community Complement dependent lymphocytotoxicity (CDC) assay is a well-known cell based assay for detecting complement activating Abs with both IgG and IgM isotypes Although the CDC assay captures most clinically relevant antibodies, it cannot detect low titer complement activating antibodies Because C1q, the first component of the classical complement activation pathway, cannot efficiently activate complement cascade unless it binds two activated Fc regions of bound antibodies in close proximity This limitation can be overcome by using Anti-Human Globulin (AHG), a bridge between anti-HLA antibody and C1q to augment CDC assay The read out of the CDC assay is based on counting dead cells caused by MAC The addition of the flow cytometry crossmatch (FCXM) in transplant laboratories has improved the sensitivity and specificity of the XM testing It has been demonstrated that positive FCXM in the presence of negative CDC or negative AHG-augmented XM can be a predictor of graft rejection 1,2 The United Network of Organ Sharing guidelines state that the CDC assay can be used for initial crossmatching, but more sensitive tests should be performed to identify patients with antibodies 3 Although FCXM is a very sensitive method, it cannot distinguish between complement and noncomplement activating antibodies The CDC assay was the only established method for detecting complement activating antibodies until Chen et al introduced C1q assay in transplantation laboratory practice C1q assay is a bead based assay that measures the first component of classical pathway of the complement activation detected in Luminex platform An initial study of the C1q assay revealed that CDC testing identifies only about 19% of complement activating Ab detected by C1q assay and only 47% of Ab detected by Luminex IgG assay 4 A study by Lachmann et al reported that C1q- and C4d-Luminex assays identified three times more specificities than the CDC assay 5 Both groups suggested that the C1q Luminex assay might enable the better prediction of the pathogenicity of anti-HLA Abs both pre- and post-transplantation 4,5 Recently, Loupy et al published an article entitled "Complement- binding Anti-HLA Antibodies and Kidney Allograft Survival " The retrospective study was conducted in a large population group to determine whether assessment for the presence of C1q- binding donor specific antibody (DSA) after transplantation might improve risk stratification for kidney-allograft loss The authors reported that patients with de novo complement binding DSA had the lowest five-year graft survival (54%) compared with patients with non-complement binding DSA (93%) and patients without DSA (94%) The presence of complement binding DSA was associated with a risk of graft loss, increased rate of AMR and increased deposition of C4d within graft capillaries The authors also demonstrated that the addition of complement binding DSA to the reference model adequately reclassified patients at lower and higher risk for graft loss 6 Although the C1q assay can be sensitive and specific, its prognostic utility for routine care of pre- and post-transplant patients is still widely debated 7-11 While, some groups have suggested that detection of C1q binding by DSA should be incorporated as part of the protocol in clinical trials of treatment and outcomes in antibody mediated allograft injury, 6,7 other groups disagree arguing that routinely implementing the C1q assay in current practice will add a burden of extra testing which may be difficult to interpret, as is sometimes true between Luminex IgG Mean Fluorescence Intensity (MFI) and FCXM results 8,9 The strength of DSA in Luminex IgG could be a strong predictor of complement fixation, 7-10 however, some studies demonstrated the lack of correlation for MFI values between Luminex IgG and C1q assays 4,5 The Loupy et al study raised questions when about half of patients with positive pre-transplant C1q-binding DSA become negative after transplantation 6 Although those patients fell into the low risk of graft loss group in post-transplant monitoring, the pre-transplant assessment of C1q binding was of limited value for predicting the course after transplantation 11 Some of the pre and post-transplant C1q changes may be due to antibody isotype and class switching Since C1q is the earliest step of complement activation, the downstream steps in the cascade could be inhibited before inducing terminal products required for MAC In addition to natural inhibitors of the early steps of the classical complement pathway, like C1q inhibitor (chondroitin-4 sulfate proteoglycan), some proteins released during pathological events inhibit C1q activation For example, heme (iron protoporphyrin IX), released from hemoglobin, myoglobin, and cytochromes due to tissue damage and hemolysis, is an endogenous negative regulator of the classical complement activation pathway at the level of C1q 12 Unconjugated bilirubin found at high serum concentration in liver diseases or hemolytic anemia as well as a factor found in rheumatoid arthritis binds to C1q and inhibits the classical complement pathway 13-15 If this is the case and the transplant center lists C1q positive Abs as unacceptable (UA) in UNet, it could limit the donor pool for sensitized transplant candidates In contrast, pre-transplant C1q negative patients could develop de novo C1q DSA, causing AMR and transplant glomerulopathy 16,17 Pre-transplant the C1q binding assay may not add value over a sensitive FCXM in predicting subsequent AMR 16 Only the classical pathway activates C1q Generally, immunoglobulins are thought to exclusively activate the classical pathway when they bind to antigen However, recent studies show that certain immunoglobulins, such as IgG-G0 glycoform, auto- Abs and IgA, can activate both lectin and alternative pathways of the complement system, which can cause autoimmune diseases and IgA nephropathy 18 The C1q assay does not detect non-HLA Abs, which may be pathogenic through non-complement fixing mechanisms, nor does it detect the presence of multiple low- titer DSAs that individually do not fix complement 7 C1q assay may miss some potentially harmful Abs that could activate the complement system by lectin or alternative pathways Although some limitations could exist in pre-transplant C1q binding assay, it is a useful test to monitor DSAs and AMR after transplantation, especially in patients receiving high dose IVIG, who cannot be monitored by IgG because of the high background artifact from the IVIG C1q assay is an informative source to limit