Specimen Handling and Measurement of Complement Biomarkers
Specimen Handling and Measurement of Complement Biomarkers
Objectives Recent studies have shown that complement hyperactivation contributes to development of thrombotic microangiopathy. The evaluation of complement biomarkers is known to be influenced by inappropriate specimen handling. However, there has been no study fully addressing this topic.
Methods Blood from each donor was subjected to 62 different handling conditions prior to complement assays.
Results Complement biomarkers (C4d/C3a/factor Bb/C5a/C5b-9) are stable at room temperature (RT) for up to 4 hours in whole blood containing citrate or EDTA. However, under similar conditions, levels of C4d and C3a were significantly higher in serum than those in plasma. Thawing of the samples on ice or at RT had no significant effect on complement levels. In contrast, thawing at 37°C resulted in striking increases in levels of the complement system in serum and citrated plasma but not in EDTA plasma. Up to four freeze/thaw cycles on ice or RT did not substantially increase the levels of C3a, factor Bb, C5a, and C5b-9 but had a significant effect on C4d. Long-term storage of citrated plasma at −80°C for up to 6 years had no significant effect on levels of complement factors.
Conclusions The results from this study thus provide crucial guidelines for future investigations using complement biomarkers to define the role of complement system in disease.
Complement dysregulation is known to contribute to a wide range of disease processes. Recent studies strongly suggest that complement hyperactivation plays an essential role in the development of various clinical phenotypes of thrombotic microangiopathies (TMAs). Atypical hemolytic uremic syndrome (aHUS) is a type of TMA caused by excessive complement activation secondary to either an inherited defect and/or acquired antibody impairing the regulatory function of the complement system. The genetic abnormalities in patients with aHUS may involve regulators of complement-alternative pathways such as factor H, factor I, or complement activation factors. A specific complement inhibitor, eculizumab, has been approved by the US Food and Drug Administration for the treatment of aHUS. New studies also have shown that complement activation biomarkers are elevated in patients with another type of TMA, thrombotic thrombocytopenic purpura (TTP). The severity of increases in complement biomarker levels appears related to worse clinical outcomes in TTP. Involvement of the complement system in TTP's disease course is also supported by a report that a patient with TTP refractory to plasma exchange therapy achieved long-term remission after being treated with a complement inhibitor (anti-C5). Furthermore, in patients with hematopoietic stem cell transplant (HSCT)–associated TMA, a study has shown a high prevalence of factor H–related gene deletion and factor H autoantibodies, suggesting that dysregulation of the complement-alternative pathway may contribute to the pathogenesis of post-HSCT TMA. Taken together, these studies support the role of complement dysfunction in the development of TMA and provide a basis for using complement biomarkers clinically to evaluate patients with TMA.
Assays for C3, C4, and CH50 have been used routinely for evaluation of complement pathways in clinical laboratories. However, these tests are generally not sensitive to the alterations in the complement system that occur in many diseases. For instance, in patients with aHUS and evidence of a genetic mutation, less than 50% show abnormal results when tested for C3, C4, and CH50 levels, and consequently, these complement levels are not routinely used to diagnose, monitor, and manage patients with aHUS. We hypothesize that the downstream proteolytic fragments of the complement system are more sensitive to subtle dysregulation in the complement pathway. These candidate downstream biomarkers include C3a, C4d, terminal pathway activation product C5b-9 and C5a, and a product of the amplification loop, complement factor Bb. We and others have recently evaluated the performance of these downstream complement biomarkers and provided supporting evidence for their application in clinical evaluation of various types of TMAs.
The analysis of complement biomarker levels in research studies and in the clinic is complex and subject to significant inaccuracies due in part to variation in sample collection and handling. Despite the considerable concern for potential false-positive results in the tests for complement biomarkers, there has been a lack of studies fully investigating preanalytical effects on complement biomarker assays. In this study, we performed a detailed evaluation of how specimen sampling, processing, and storage conditions affect the results of biomarker assays for complement activation products C3a, C4d, C5a, C5b-9, and factor Bb. The objective of the study is to provide guidelines for clinical and research laboratories with regard to the handling of specimens for complement assays and ultimately to ensure that more accurate and reliable complement assays are being used in both clinical and research evaluation of patients with abnormal complement functions.
Abstract and Introduction
Abstract
Objectives Recent studies have shown that complement hyperactivation contributes to development of thrombotic microangiopathy. The evaluation of complement biomarkers is known to be influenced by inappropriate specimen handling. However, there has been no study fully addressing this topic.
Methods Blood from each donor was subjected to 62 different handling conditions prior to complement assays.
Results Complement biomarkers (C4d/C3a/factor Bb/C5a/C5b-9) are stable at room temperature (RT) for up to 4 hours in whole blood containing citrate or EDTA. However, under similar conditions, levels of C4d and C3a were significantly higher in serum than those in plasma. Thawing of the samples on ice or at RT had no significant effect on complement levels. In contrast, thawing at 37°C resulted in striking increases in levels of the complement system in serum and citrated plasma but not in EDTA plasma. Up to four freeze/thaw cycles on ice or RT did not substantially increase the levels of C3a, factor Bb, C5a, and C5b-9 but had a significant effect on C4d. Long-term storage of citrated plasma at −80°C for up to 6 years had no significant effect on levels of complement factors.
Conclusions The results from this study thus provide crucial guidelines for future investigations using complement biomarkers to define the role of complement system in disease.
Introduction
Complement dysregulation is known to contribute to a wide range of disease processes. Recent studies strongly suggest that complement hyperactivation plays an essential role in the development of various clinical phenotypes of thrombotic microangiopathies (TMAs). Atypical hemolytic uremic syndrome (aHUS) is a type of TMA caused by excessive complement activation secondary to either an inherited defect and/or acquired antibody impairing the regulatory function of the complement system. The genetic abnormalities in patients with aHUS may involve regulators of complement-alternative pathways such as factor H, factor I, or complement activation factors. A specific complement inhibitor, eculizumab, has been approved by the US Food and Drug Administration for the treatment of aHUS. New studies also have shown that complement activation biomarkers are elevated in patients with another type of TMA, thrombotic thrombocytopenic purpura (TTP). The severity of increases in complement biomarker levels appears related to worse clinical outcomes in TTP. Involvement of the complement system in TTP's disease course is also supported by a report that a patient with TTP refractory to plasma exchange therapy achieved long-term remission after being treated with a complement inhibitor (anti-C5). Furthermore, in patients with hematopoietic stem cell transplant (HSCT)–associated TMA, a study has shown a high prevalence of factor H–related gene deletion and factor H autoantibodies, suggesting that dysregulation of the complement-alternative pathway may contribute to the pathogenesis of post-HSCT TMA. Taken together, these studies support the role of complement dysfunction in the development of TMA and provide a basis for using complement biomarkers clinically to evaluate patients with TMA.
Assays for C3, C4, and CH50 have been used routinely for evaluation of complement pathways in clinical laboratories. However, these tests are generally not sensitive to the alterations in the complement system that occur in many diseases. For instance, in patients with aHUS and evidence of a genetic mutation, less than 50% show abnormal results when tested for C3, C4, and CH50 levels, and consequently, these complement levels are not routinely used to diagnose, monitor, and manage patients with aHUS. We hypothesize that the downstream proteolytic fragments of the complement system are more sensitive to subtle dysregulation in the complement pathway. These candidate downstream biomarkers include C3a, C4d, terminal pathway activation product C5b-9 and C5a, and a product of the amplification loop, complement factor Bb. We and others have recently evaluated the performance of these downstream complement biomarkers and provided supporting evidence for their application in clinical evaluation of various types of TMAs.
The analysis of complement biomarker levels in research studies and in the clinic is complex and subject to significant inaccuracies due in part to variation in sample collection and handling. Despite the considerable concern for potential false-positive results in the tests for complement biomarkers, there has been a lack of studies fully investigating preanalytical effects on complement biomarker assays. In this study, we performed a detailed evaluation of how specimen sampling, processing, and storage conditions affect the results of biomarker assays for complement activation products C3a, C4d, C5a, C5b-9, and factor Bb. The objective of the study is to provide guidelines for clinical and research laboratories with regard to the handling of specimens for complement assays and ultimately to ensure that more accurate and reliable complement assays are being used in both clinical and research evaluation of patients with abnormal complement functions.
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