Table 7. Studies describing assay... | American Society of Hematology

Table 7.

Studies describing assay systems that can detect cellular/surface directed complement activation

First author/reference	Setup of test	Main finding	Other interesting findings	Comment
Noris⁶⁶	Cultured and stimulated HMEC-1 are exposed to patient sera; C5b-9 (or MAC) deposition is detected	• Identifies cell surface-restricted C activation in serum of aHUS patients in acute disease or remission (supporting the second hit theory) • Monitors effectiveness of C inhibition in patients in an ex vivo assay	• aHUS serum regularly leads to cell-surface C5b-9 deposition, whereas C3G sera do not • Stimulation of HMEC-1 cells with ADP, LPS or thrombin is equally effective to sensitize cells for complement deposition	These assays are not expected to detect aHUS associated with alterations in DGKE, MCP or THBD genes or function
Gavriilaki¹⁷⁴	Engineered PIG-A-null reagent cell line is exposed to patient sera and viability (or insertion of C5b-9) is detected	• Identifies cell surface-restricted C activation in serum of aHUS patients in acute disease or remission (supporting the second hit theory) • Monitors effectiveness of C inhibition in patients in an ex vivo assay	• Test was shown to distinguish aHUS from other TMAs like TTP • Test setup was shown to identify APS patients at higher risk to develop thrombotic events¹⁷²	These assays are not expected to detect aHUS associated with alterations in DGKE, MCP or THBD genes or function
Rand¹⁷⁰	Phospholipid vesicles pre-incubated with APS plasmas (1st step) are analyzed for C5b-9 insertion when exposed to NHS (2nd step).	Distinguishes APS from other inflammatory and thrombotic disorders not associated with aPL-abs	Plasma levels of C5a and sC5b-9 were higher in APS patients but not significantly increased when compared with healthy controls	Artificially produced PL vesicles appear especially suitable and are easily available
Anliker⁸⁸	RBCs of patients with a PNH III RBC clone >20% exposed to plasma with allo- or autoantibodies (first step) and analyzed for MAC mediated hemolysis in NHS (second step)	• The potency of 174 different patient-derived RBC antibodies to activate complement was correlated to their antigen specificity • Allows to identify antibodies that likely inflict complement-mediated hemolysis	The C5 inhibition abrogated mild but not strong complement TP activation in vitro. However, residual TP activity could be further depressed by combining C5 inhibitors with proximal C inhibitors	Limiting applicability: ABO matched reagents (or RBCs with ABO ‘O’) are needed; PNH RBCs can be frozen, but supply of PNH RBCs is sparse

First author/reference	Setup of test	Main finding	Other interesting findings	Comment
Noris⁶⁶	Cultured and stimulated HMEC-1 are exposed to patient sera; C5b-9 (or MAC) deposition is detected	• Identifies cell surface-restricted C activation in serum of aHUS patients in acute disease or remission (supporting the second hit theory) • Monitors effectiveness of C inhibition in patients in an ex vivo assay	• aHUS serum regularly leads to cell-surface C5b-9 deposition, whereas C3G sera do not • Stimulation of HMEC-1 cells with ADP, LPS or thrombin is equally effective to sensitize cells for complement deposition	These assays are not expected to detect aHUS associated with alterations in DGKE, MCP or THBD genes or function
Gavriilaki¹⁷⁴	Engineered PIG-A-null reagent cell line is exposed to patient sera and viability (or insertion of C5b-9) is detected	• Identifies cell surface-restricted C activation in serum of aHUS patients in acute disease or remission (supporting the second hit theory) • Monitors effectiveness of C inhibition in patients in an ex vivo assay	• Test was shown to distinguish aHUS from other TMAs like TTP • Test setup was shown to identify APS patients at higher risk to develop thrombotic events¹⁷²	These assays are not expected to detect aHUS associated with alterations in DGKE, MCP or THBD genes or function
Rand¹⁷⁰	Phospholipid vesicles pre-incubated with APS plasmas (1st step) are analyzed for C5b-9 insertion when exposed to NHS (2nd step).	Distinguishes APS from other inflammatory and thrombotic disorders not associated with aPL-abs	Plasma levels of C5a and sC5b-9 were higher in APS patients but not significantly increased when compared with healthy controls	Artificially produced PL vesicles appear especially suitable and are easily available
Anliker⁸⁸	RBCs of patients with a PNH III RBC clone >20% exposed to plasma with allo- or autoantibodies (first step) and analyzed for MAC mediated hemolysis in NHS (second step)	• The potency of 174 different patient-derived RBC antibodies to activate complement was correlated to their antigen specificity • Allows to identify antibodies that likely inflict complement-mediated hemolysis	The C5 inhibition abrogated mild but not strong complement TP activation in vitro. However, residual TP activity could be further depressed by combining C5 inhibitors with proximal C inhibitors	Limiting applicability: ABO matched reagents (or RBCs with ABO ‘O’) are needed; PNH RBCs can be frozen, but supply of PNH RBCs is sparse

C, complement; DGKE, diacylglycerolkinase-ɛ; HMEC-1, human microvascular endothelial cells; MCP, membrane cofactor protein or CD46; THBD, thrombomodulin; TTP, thrombotic thrombocytopenic purpura.

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