Thrombotic Microangiopathy (aHUS and TTP)

In patients with atypical HUS and other forms of thrombotic microangiopathy in which complement activation plays a role, a diagnostic assessment of complement activation can serve as a useful guide of therapeutic interventions such as eculizumab.

Eculizumab is a monoclonal antibody directed against complement protein C5, and shuts down complement-mediated thrombotic microangiopathy in such patients. 

Markers of complement activation such as C3a, C5a, Bb and soluble C5b-9 (sMAC or sTCC) and functional complement testing such as CH50 can provide valuable insight as to the presence and severity of complement activation, as well as the reversal of this activation with eculizumab. 

The HUS Complement Activation Panel includes C3a, C5a, Bb, sC5b-9, CH50, and sMCP.

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The eculizumab level (free anti-C5) is a measurement of the serum level of eculizumab (Soliris, Alexion Pharmaceuticals), a humanized IgG₄ monoclonal antibody directed against the complement protein C5.  By binding to C5, eculizumab prevents cleavage of C5 to its active fragments C5a and C5b, thereby preventing formation of C5b-9 and halting the process of complement-mediated cell destruction.  

Adequate dosage and dosing interval of eculizumab result in a complete blockade of activation of the terminal complement pathway.  If a patient is not being adequately dosed with eculizumab, breakthrough activation of the complement system and subsequently disease reactivation may occur.  Along with clinical markers of disease activity and other laboratory measures of terminal complement pathway activity (such as CH50, C5a and sC5b-9), the eculizumab level may be used to guide therapeutic decision making as to adequate dosage and dosing interval. 

Reference interval: Eculizumab is considered to be present in a therapeutic level in aHUS if the trough serum concentration is >99µg/mL (Legendre CM et al, NEJM 2013), and in PNH if the serum concentration is >35µg/mL (Hillmen P et al, NEJM 2004).  

Synonyms: Soliris, free anti-C5 

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The evaluation of thrombotic microangiopathy includes testing the regulatory components of the alternative complement pathway for aHUS and the activity of ADAMTS13. 

Mutations in proteins which regulate complement activation, as well as autoantibodies that neutralize the function of these proteins, are associated with aHUS. 

In many patients, mutations in these proteins lead to quantitative deficiencies of one or more of these proteins.

The TMA (aHUS and TTP) Diagnostic Panel performed by our clinical laboratories tests the quantitative levels of C3, C4, Factor H, Factor I, Factor B; ADAMTS13 activity; autoantibody against Factor H; and flow cytometry of Membrane Cofactor Protein / CD46 expression on leukocytes.

• TMA (aHUS and TTP) Genetic Testing Information Sheet
• TMA diagnostic testing algorithm
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Mutations in proteins which regulate complement activation, as well as autoantibodies that neutralize the function of these proteins, are associated with aHUS. 

Loss of function mutations in complement regulatory proteins Factor H (CFH), Factor H-related 5 (CFHR5), Factor I (CFI), Membrane Cofactor Protein / CD46 (CD46), and Thrombomodulin (THBD), as well as gain of function mutations in alternative pathway components Factor B (CFB) and C3 (C3) have all been identified in patients with atypical HUS. 

Mutational analysis by sequencing of the 10 known genes associated with aHUS (CFH, CFI, MCP, C3, CFB, THBD, CFHR5, CFHR1, CFHR3, and most recently identified DGKE) provides the most comprehensive assessment of defects in complement regulation. 

This informs not only the disease process as likely complement mediated, but also provides therapeutic and prognostic information in patients with aHUS with regards to risk of progression to end-stage renal disease, risk of relapse, and risk of recurrence in kidney transplant.

• TMA (aHUS and TTP) Genetic Testing Information Sheet
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ADAMTS13 Activity

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ADAMTS13 is a serum protease that cleaves ultra large multimers of von Willebrand Factor (vWF) into smaller oligomeric subunits, thus regulating the interaction of platelets with vWF in the microvasculature.  Absent or severely deficient (<10%) ADAMTS13 activity allows formation of platelet microthrombi, which in turn obstructs arterioles and capillaries, generating the clinical sequelae of TTP.  ADAMTS13 activity is measured by Fluorescence Resonance Energy Transfer (FRET) using the substrate FRETS-VWF73 (Kokame et al, Br J Haematol. 2005 Apr;129(1):93-100).

ADAMTS13 Inhibition Test

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In patients with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 Activity may be severely decreased (<10%) in the presence of an inhibitor, typically an IgG autoantibody, but may also be severely decreased due to mutations in the ADAMTS13 gene leading to a quantitative or qualitative defect of the ADAMTS13 protein.  To separate these entities, the presence of an inhibitor is determined by measuring the ability of heat-treated patient plasma to inhibit ADAMTS13 Activity as measured by FRET in normal plasma; inhibitor activity is expressed as percent inhibition of the ADAMTS13 Activity of pooled normal plasma.  In this assay, 50% inhibition is roughly equivalent to 1.0 Bethesda unit/mL.  Strong antibody-mediated inhibition of ADAMTS13 activity (>90%) is seen in approximately half of the patients with acquired or idiopathic TTP.  The significance of milder ADAMTS13 inhibition is unclear, particularly when the overall ADAMTS13 level is not severely decreased. The assay used does not distinguish between antibody-mediated and non-specific ADAMTS13 inhibition.

ADAMTS13 Inhibiting Antibody

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Autoantibodies (typically IgG) that neutralize or enhance clearance of ADAMTS13 may severely inhibit ADAMTS13 activity and therefore allow the accumulation of ultra large multimers of vWF in plasma, and ultimately adhesion to platelets in the microvasculature leading to the clinical sequelae of thrombotic thrombocytopenic purpura (TTP).  These autoantibodies are believed to be the major cause for idiopathic TTP.  This test detects and quantifies autoantibodies against ADAMTS13 by ELISA.  Patients with idiopathic TTP usually require therapeutic plasma exchange to achieve clinical remission. Persistence of ADAMTS13 deficiency or an inhibitor/antibody during clinical remission suggests an increased risk for recurrence of symptomatic TTP.   

ADAMTS13 Gene Sequencing

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ADAMTS13 Panel

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ADAMTS13 Testing Algorithm

This panel testing ADAMTS13 Activity utilizes a reflexive testing algorithm. ADAMTS13 Activity is always performed by Fluorescence Resonance Energy Transfer (FRET).  If the ADAMTS13 Activity is less than or equal to 30%, the ADAMTS13 Inhibition Test is performed.  If the amount of ADAMTS13 Inhibition is found to be greater than 30%,  the ADAMTS13 Inhibiting Autoantibody is performed.  Thrombotic thrombocytopenic purpura (TTP) is generally characterized by an ADAMTS13 Activity <10%.

aHUS Complement Activation Panel

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Includes the following:

• CH50
• SC5b-9 / MAC
• C3a
• C5a
• Bb

Bb (Bb Fragment of Factor B) (ELISA)

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The Bb fragment of Factor B provides evidence of activation of the alternative complement pathway. Factor B, when bound to C3b, is cleaved by Factor D to yield fragments Ba and Bb and the resulting complex C3bBb is the alternative pathway C3 convertase.  This complex is capable of cleaving additional C3 or forming a C5 convertase, cleaving C5 into its active fragments, C5a and C5b-9 (membrane attack complex). Failure to regulate the activation of this pathway has been associated with complement-mediated diseases including atypical hemolytic uremic syndrome and membranoproliferative glomerulonephritis type II/dense deposit disease.  Since the Bb molecule is unique to the alternative pathway of complement, elevated levels of Bb as measured by ELISA may provide a marker for complement activation via this pathway.

C3 Level (Nephelometry)

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The complement system can be activated via three reaction pathways: the classical pathway, which is triggered primarily by cell-bound immune complexes; the mannan-binding lectin pathway, which is triggered by specific carbohydrate groups on microorganisms; and the alternative pathway, which is activated constitutively on all cell surfaces especially microorganisms.  The complement component C3 is a key protein in all three reaction pathways, and complement activation is associated with consumption of C3 and a reduction in serum concentration.  Diminished serum concentrations of C3 is observed through activation of the alternative pathway, and may be seen in atypical hemolytic uremic syndrome (aHUS) and forms of membranoproliferative glomerulonephritis (MPGN). Diminished serum concentrations of C3 is also observed through activation of the classical pathway (typically in combination with diminished serum levels of complement component C4) in active systemic lupus erythematosus (SLE), in some forms of membranoproliferative glomerulonephritis and in other immune complex diseases.

C3 Gene Sequencing

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C3a

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Activation of the classical, alternative or lectin complement pathways results in the formation of a C3 convertase multimolecular enzyme capable of cleaving C3 to C3a and C3b. C3a has been shown to increase vascular permeability, to be spasmogenic and chemotactic, and to induce the release of pharmacologically active mediators from a number of cell types.

C4 Level (Nephelometry)

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Diminished serum concentrations of C4 is observed primarily in activation of the classical pathway by immune complexes such as in active systemic lupus erythematosus (SLE), in forms of membranoproliferative glomerulonephritis (MPGN), and in other immune complex diseases (e.g. “shunt nephritis” and serum sickness), and is useful in distinguishing systemic activation of the classical pathway versus activation of the alternative complement pathway as seen in atypical hemolytic uremic syndrome (aHUS) and dense deposit disease/MPGN type II.

C5a

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Activation of the classical, alternative, or lectin complement pathways results in the formation of a C5 convertase multimolecular enzyme capable of cleaving C5 to C5a and C5b. C5a has a host of biologic functions including mast cell degranulation, chemotaxis, leukosequestration, as well as cellular activation via binding to the C5a receptor.

CD46 (MCP) Gene Sequencing

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CD46 / Membrane Cofactor Protein (MCP) Expression 

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Atypical hemolytic uremic syndrome (aHUS) is associated with defects in the regulation of the alternative complement pathway and results in microvascular injury. Evaluation of the membrane-bound complement proteins – or Membrane Cofactor Protein (MCP) – can be evaluated by testing the CD46 expression on white blood cells (WBC) using flow cytometry. CD46 is normally expressed on all WBC lineages. Mutations of the MCP gene are associated with aHUS and may lead to decreased CD46 expression on the surface of cells including WBCs. 

CFB Gene Sequencing

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CFH Gene Sequencing  

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CFHR1 Gene Sequencing

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CFHR3 Gene Sequencing

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CFHR3-CFHR1 Deletion Analysis

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CFHR5 Gene Sequencing

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CFI Gene Sequencing

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CH50 (Hemolytic Method)

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The traditional method for determination of functional complement activity is the total hemolytic (CH50) assay.  This assay measures the ability of the test sample to lyse a standardized suspension of sheep erythrocytes coated with anti-sheep antibody.  Activation of both the classical and terminal complement pathways are measured in this reaction.  A normal CH50 result is dependent on the presence and functionality of both classical pathway (C1q, C4, C2, C3) and terminal (C5, C6, C7, C8, and C9) complement components, and is abnormally low if any component is defective. 

More recently, this test has become highly useful to monitor patients with either atypical hemolytic uremic syndrome (aHUS) or other forms of thrombotic microangiopathy (TMA) being treated with eculizumab, a monoclonal antibody directed against C5.  Adequate dosage and dosing interval of eculizumab should result in a total blockade of activation of the terminal complement pathway, and a patient’s CH50 should be at or near 0 as a result.  If a patient is not being adequately dosed with eculizumab, breakthrough activation of the complement system and subsequently disease reactivation may occur.

DGKE Gene Sequencing

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Factor B Level (Radial immunodiffusion)

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Factor B (CFB) is a single-chain glycoprotein which provides the catalytic subunit of the C3/C5 convertases of the alternative complement pathway. Assembly of the C3 convertase (C3bBb) requires binding of Factor B to C3b followed by cleavage to Bb mediated by Factor D.  This C3 convertase provides a positive amplification loop for the classical and alternative complement pathways.  Gain of function mutations in Factor B causing enhanced binding to C3b and have been associated with atypical hemolytic uremic syndrome (aHUS).  Due to consumption by formation of C3 convertase, serum levels of intact Factor B may be decreased in aHUS.  The Bb fragment of Factor B is the serine protease element of this convertase, and elevations of Factor Bb may be regarded as an indicator of the alternative pathway of complement activation.

Factor H Level (Radial Immunodiffusion)

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Factor H (CFH) is a regulatory protein of the alternative pathway of the complement system. Factor H is a serum glycoprotein that binds to both C3b and sialic acid/glycosaminoglycans, which enables it to act as co-factor for the Factor I-mediated proteolytic inactivation of C3b and accelerate the decay of the alternative pathway C3-convertase (C3bBb), thus regulating complement activation both in the fluid phase and on cellular surfaces.  Decreased serum levels of Factor H due to mutations in Factor H or autoantibodies against Factor H may lead to subsequent unregulated formation of the alternative pathway C3 convertase C3bBb, and can be seen in some patients with aHUS as well as dense deposit disease/ membranoproliferative glomerulonephritis (MPGN) type II/Dense Deposit Disease.

Factor H Autoantibody (ELISA)

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Factor H is a complement regulatory protein, which main function is to bind to C3b deposited on the surface of endothelial cells and facilitate its cleavage to an inactive form, iC3b, by Factor I.  Failure to facilitate this cleavage lead to unregulated formation of the C3 convertase C3bBb on the surface of the cell, and ultimately unregulated formation of the membrane attack complex C5b-9 on the cell surface leading to cell lysis.  Inhibitory autoantibodies directed against Factor H may reduce serum levels of Factor H and dysregulate the alternative pathway of the complement system, and have been identified in ~5% of patients with atypical hemolytic uremic syndrome (aHUS).  Removal of anti-factor H antibodies from the bloodstream by plasmapheresis or the use of immunosuppressive drugs to eliminate the autoantibody production has shown benefit in the outcome of these diseases.

Factor I Level (Radial Immunodiffusion)

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Factor I (CFI) is a serine protease that is essential for regulating the complement system by cleaving and inactivating C4b and C3b, and thus preventing the assembly of the C3 and C5 convertases.  Mutations in CFI have been associated with a predisposition to atypical hemolytic uremic syndrome (aHUS), due to a failure to inactivate C3b by cleavage to iC3b, with subsequent unregulated formation of the alternative pathway C3 convertase C3bBb and ultimately membrane attack complex C5b-9 on the endothelial cell surface leading to thrombotic microangiopathy.  Decreased serum levels of Factor I have been identified in some patients with mutations in CFI leading to aHUS.

Familial Thrombotic Thrombocytopenic Purpura

• ADAMTS13 panel
• ADAMTS13 activity
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MCP Gene Sequencing

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SC5b-9 (MAC)

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SC5b-9 / MAC: Soluble Terminal Complement Complex / Membrane Attack Complex 

The SC5b-9 enzyme immunoassay (EIA) measures the amount of the SC5b-9 complex present in human plasma.  The Terminal Complement Complex (TCC, SC5b-9) is generated by the assembly of C5 through C9 as a consequence of activation of the complement system by either the classical, lectin or alternative pathway.  The membrane attack complex (MAC), a form of TCC, is a stable complex that mediates the irreversible target cell membrane damage associated with complement activation.  Complexes formed in the absence of a target membrane bind to naturally occurring regulatory serum proteins, e.g. the S protein forming non-cytolytic complexes in plasma.  The SC5b-9 EIA test quantitates SC5b-9 in plasma.  

The drug eculizumab, a terminal complement inhibitor, has been shown to inhibit hemolysis in patients with paroxysmal nocturnal hemoglobinuria (PNH) and to inhibit complement-mediated thrombotic microangiopathy in patients with atypical HUS (aHUS). Eculizumab is a humanized monoclonal antibody that binds to the complement protein C5 with high affinity, thereby inhibiting its cleavage to C5a and C5b and preventing the generation of the cytolytic terminal complement complex, C5b-9.  Elevated plasma levels of SC5b-9 may be an indicator of an insufficient level of eculizumab to maintain blocking the formation of the terminal attack complex.

THBD Gene Sequencing

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Thrombotic Microangiopathy (aHUS and TTP) Diagnostic Panel

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Includes the following:

• C3 Level
• C4 Level
• Factor H Level
• Factor I Level
• Factor B Level
• Factor H Autoantibody
• ADAMTS13 Activity
• Membrane Cofactor Protein / CD46 Expression

Thrombotic Thrombocytopenic Purpura (TTP) Testing 

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• ADAMTS13 Testing Panel (includes the following):
• ADAMTS13 Activity
• ADAMTS13 Antibody Level
• ADAMTS13 Inhibition Test
• ADAMTS13 gene sequencing