TMA Functional Panel
| Label Mnemonic: | TMAFP |
| Epic code: | LAB8807 |
| Downtime form: | A-1a Doctor/Provider Orders - Pathology Core and Specialty Care Nursery |
Commercial Mailout Laboratory
6240-8 RCP
356-8593
6240-8 RCP
356-8593
Specimen(s):
Serum and Plasma
Collection Medium:
 | and |  |
| Red top tube 5 mL (Clot Activator) | Pink top tube 6 mL (K2-EDTA) |
Minimum:
Preferred Minimum: 2 mL serum in red top tube and 2 mL EDTA plasma in
pink top tube
Delivery Instructions:
2-4 weeks upon receipt at reference laboratory
Interpretive Data:
Factor H Autoantibodies
Dense Deposit Disease (DDD, aka Membranoproliferative Glomerulonephritis Type II, MPGNII) Factor H autoantibodies have been associated with DDD (Meri, et al., 1992). In patients with DDD, these autoantibodies bind to and block the N-terminal region of the Factor H protein, which compromises its fluid-phase regulatory function.
Hemolytic Assay - Sheep Erythrocyte Lysis Assay
Sheep erythrocytes are non-activating surfaces. This assay measures complement-mediated lysis of sheep erythrocytes secondary to activation of the alternative pathway (AP). Lysis can be seen in the serum of patients with aHUS and DDD.
CH50 Level
Dense Deposit Disease; C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome
CH50 measures total hemolytic activity of the classical and terminal pathways using sensitized sheep erythrocytes. CH50 can be low if complement components in the classical pathway (C1, C4, C2, C3) or terminal pathway (C5 through C9) are reduced or absent. Since most patients with C3G (especially DDD) have exceedingly low plasma C3 levels, their CH50s are also typically low.
Alternative Pathway Functional Assay
The Alternative Pathway Functional Assay (APFA) measures activity of the alternative pathway (AP) of complement. Specific initiators are used to stimulate the AP on patient-derived sera; neoantigens of C9 produced as a result of terminal complement complex (C5b-9) activation are measured. Consumption or depleted of AP complement proteins will result in a low (abnormal) APFA.
Complement Bb Fragment Level Assay
The activation of alternative pathway (AP) of complement generates the active proteolytic enzyme Bb. In the presence of C3b, factor B (MW: 93 kDa) binds to C3b to form the pre-convertase (C3bB). Factor D cleaves factor B releasing Ba (MW: 33 kDa) and generating the active proteolytic enzyme Bb (MW: 66kDa). The Bb subunit is catalytically active and cleaves new C3 to C3a and C3b. C3bBb recruits additional available C3b to form the C5 convertase, C3bBbC3b, launching terminal pathway activation. C3 convertase can be dissociated by spontaneous decay or complement regulators (factor H, CR1). It can also be inactivated by factor I-mediated C3b cleavage in presence of cofactors.
The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. Plasma levels of Bb are elevated in both DDD and C3GN as compared to controls (p<0.001) consistent with dysregulation of the C3 convertase in both diseases (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).
Plasma Factor H Level
Dense Deposit Disease, C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome
Complement Factor H (FH; MW: 155 kDa) is an important fluid-phase and cell-surface regulator of alternative pathway (AP) activity. Patients with FH mutations or FH autoantibodies may have reduced plasma FH levels and/or function, and are at-risk to develop atypical hemolytic uremic syndrome or C3 glomerulopathy.
Plasma Complement Factor I Level
Atypical Hemolytic Uremic Syndrome
Complement Factor I (FI; MW: 88 kDa) is an important regulator of complement activity triggered through the classical and alternative pathways. FI limits complement activation by cleaving surface-bound and fluid-phase C3b and C4b, preventing the assembly of the C3 and C5 convertases. Patients with FI mutations may have reduced plasma FI levels and/or function, and are at-risk to develop atypical hemolytic uremic syndrome or C3 glomerulopathy.
Complement Factor B Level Assay
Factor B (MW: 93 kDa) is a complement protein unique to the alternative pathway (AP). In the presence of C3b, FB binds to C3b to form the pre-convertase (C3bB). Factor D cleaves factor B releasing Ba (MW: 33 kDa) to generate the active proteolytic enzyme Bb (MW: 66kDa). The Bb subunit is the catalytically active site of the C3bBb C3 convertase complex and cleaves new C3 to C3a and C3b. If C3bBb recruits additional available C3b, the C5 convertase, C3bBbC3b, forms launching terminal pathway activation. C3 convertase can be dissociated by spontaneous decay or complement regulators (factor H, CR1). It can also be inactivated by factor I-mediated C3b cleavage in presence of cofactors.
The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. In both DDD and C3GN, patients often have lower levels of factor B as compared to controls (p<0.001) consistent with dysregulation of the C3 convertase in both diseases (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).
Complement C3 Plasma Level Assay
Complement C3 (MW: 183 kDa) is one of the most abundant plasma proteins. It is a pivotal component of complement and is central to the activation cascades. The mature protein is composed of two disulfide-bound polypeptide chains (C3α and C3β). The three complement activation pathways (alternative, classical, lectin) converge at the stage of C3 cleavage to generate the activated form of C3, which is C3b. C3b generates new C3 convertases by interacting with factors B, D and properdin. In the presence of abundant C3b, C5 convertases are formed.
Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are two ultra-rare renal diseases characterized by fluid-phase dysregulation of C3 and C5 convertases that can lead to partial or complete consumption of circulating complement components, including C3. Consumption of C3 is consistent with activation of the alternative pathway of complement (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).
C3 levels are also reduced in 30% to 50% of patients with atypical hemolytic uremic syndrome (aHUS) carrying Factor H mutations, and 20% to 30% of patients carrying Factor I mutations, a finding consistent with complement-dependent disease (Loirat & Loirat & Frémeaux-Bacchi, 2011). Concentrations of factors H and I can clarify the mechanism of C3 consumption. In ~60% of aHUS patients, C3, Factor B, Factor H and Factor I levels are normal. In these patients, the type of complement- associated defect cannot be predicted by measuring plasma protein levels but may be discoverable by genetic analysis.
Plasma Complement C4 Level
Dense Deposit Disease, C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome
Complement C4 (MW: 188 kDa), a central complement component in the classical and lectin pathways, is required to generate C4b2a, the C3 convertase of the classical pathway. C4 is cleaved to C4a (anaphylatoxin; 8 kDa) and C4b (180 kDa). C4b binds C2, and after removal of non-catalytic domain on C2, the C4b2a complex is formed. Unlike plasma C3, plasma C4 levels are typically normal in aHUS and C3G, however this test helps to rule out other complement-mediated renal diseases.
Soluble C5b-9 Assay
The complement system consists of three initiating pathways - the classical, alternative and lectin - activation of which leads to the generation of C3 and C5 convertases. The latter initiates the sequential activation of the terminal pathway (C5 to C9) resulting in the formation of pore-forming membrane attack complex C5b-9 (MAC), a stable complex that mediates irreversible cell lysis. Fluid-phase C5b-9 complexes (soluble C5b-9) may also form in complex with regulatory proteins like protein S.
C3 Glomerulopathy (C3G): The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. Soluble C5b-9 is elevated in both DDD and C3GN, but only the difference between C3GN and controls reaches statistical significance (p<0.001 for only C3GN) (see Zhang et al., Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).
Atypical Hemolytic Uremic Syndrome: Soluble C5b-9 levels are elevated in the majority of patients with active atypical hemolytic uremic syndrome but less frequently when the disease is in remission. Soluble C5b-9 may be useful as a biomarker to monitor activity of terminal pathway of complement (Bu et al., Soluble C5b-9 as a biomarker for complement activation in atypical hemolytic uremic syndrome, AJKD 2015).
Note: ADAMTS-13 Activity Assay is available 'a la carte'.
Dense Deposit Disease (DDD, aka Membranoproliferative Glomerulonephritis Type II, MPGNII) Factor H autoantibodies have been associated with DDD (Meri, et al., 1992). In patients with DDD, these autoantibodies bind to and block the N-terminal region of the Factor H protein, which compromises its fluid-phase regulatory function.
Hemolytic Assay - Sheep Erythrocyte Lysis Assay
Sheep erythrocytes are non-activating surfaces. This assay measures complement-mediated lysis of sheep erythrocytes secondary to activation of the alternative pathway (AP). Lysis can be seen in the serum of patients with aHUS and DDD.
CH50 Level
Dense Deposit Disease; C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome
CH50 measures total hemolytic activity of the classical and terminal pathways using sensitized sheep erythrocytes. CH50 can be low if complement components in the classical pathway (C1, C4, C2, C3) or terminal pathway (C5 through C9) are reduced or absent. Since most patients with C3G (especially DDD) have exceedingly low plasma C3 levels, their CH50s are also typically low.
Alternative Pathway Functional Assay
The Alternative Pathway Functional Assay (APFA) measures activity of the alternative pathway (AP) of complement. Specific initiators are used to stimulate the AP on patient-derived sera; neoantigens of C9 produced as a result of terminal complement complex (C5b-9) activation are measured. Consumption or depleted of AP complement proteins will result in a low (abnormal) APFA.
Complement Bb Fragment Level Assay
The activation of alternative pathway (AP) of complement generates the active proteolytic enzyme Bb. In the presence of C3b, factor B (MW: 93 kDa) binds to C3b to form the pre-convertase (C3bB). Factor D cleaves factor B releasing Ba (MW: 33 kDa) and generating the active proteolytic enzyme Bb (MW: 66kDa). The Bb subunit is catalytically active and cleaves new C3 to C3a and C3b. C3bBb recruits additional available C3b to form the C5 convertase, C3bBbC3b, launching terminal pathway activation. C3 convertase can be dissociated by spontaneous decay or complement regulators (factor H, CR1). It can also be inactivated by factor I-mediated C3b cleavage in presence of cofactors.
The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. Plasma levels of Bb are elevated in both DDD and C3GN as compared to controls (p<0.001) consistent with dysregulation of the C3 convertase in both diseases (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).
Plasma Factor H Level
Dense Deposit Disease, C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome
Complement Factor H (FH; MW: 155 kDa) is an important fluid-phase and cell-surface regulator of alternative pathway (AP) activity. Patients with FH mutations or FH autoantibodies may have reduced plasma FH levels and/or function, and are at-risk to develop atypical hemolytic uremic syndrome or C3 glomerulopathy.
Plasma Complement Factor I Level
Atypical Hemolytic Uremic Syndrome
Complement Factor I (FI; MW: 88 kDa) is an important regulator of complement activity triggered through the classical and alternative pathways. FI limits complement activation by cleaving surface-bound and fluid-phase C3b and C4b, preventing the assembly of the C3 and C5 convertases. Patients with FI mutations may have reduced plasma FI levels and/or function, and are at-risk to develop atypical hemolytic uremic syndrome or C3 glomerulopathy.
Complement Factor B Level Assay
Factor B (MW: 93 kDa) is a complement protein unique to the alternative pathway (AP). In the presence of C3b, FB binds to C3b to form the pre-convertase (C3bB). Factor D cleaves factor B releasing Ba (MW: 33 kDa) to generate the active proteolytic enzyme Bb (MW: 66kDa). The Bb subunit is the catalytically active site of the C3bBb C3 convertase complex and cleaves new C3 to C3a and C3b. If C3bBb recruits additional available C3b, the C5 convertase, C3bBbC3b, forms launching terminal pathway activation. C3 convertase can be dissociated by spontaneous decay or complement regulators (factor H, CR1). It can also be inactivated by factor I-mediated C3b cleavage in presence of cofactors.
The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. In both DDD and C3GN, patients often have lower levels of factor B as compared to controls (p<0.001) consistent with dysregulation of the C3 convertase in both diseases (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).
Complement C3 Plasma Level Assay
Complement C3 (MW: 183 kDa) is one of the most abundant plasma proteins. It is a pivotal component of complement and is central to the activation cascades. The mature protein is composed of two disulfide-bound polypeptide chains (C3α and C3β). The three complement activation pathways (alternative, classical, lectin) converge at the stage of C3 cleavage to generate the activated form of C3, which is C3b. C3b generates new C3 convertases by interacting with factors B, D and properdin. In the presence of abundant C3b, C5 convertases are formed.
Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are two ultra-rare renal diseases characterized by fluid-phase dysregulation of C3 and C5 convertases that can lead to partial or complete consumption of circulating complement components, including C3. Consumption of C3 is consistent with activation of the alternative pathway of complement (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).
C3 levels are also reduced in 30% to 50% of patients with atypical hemolytic uremic syndrome (aHUS) carrying Factor H mutations, and 20% to 30% of patients carrying Factor I mutations, a finding consistent with complement-dependent disease (Loirat & Loirat & Frémeaux-Bacchi, 2011). Concentrations of factors H and I can clarify the mechanism of C3 consumption. In ~60% of aHUS patients, C3, Factor B, Factor H and Factor I levels are normal. In these patients, the type of complement- associated defect cannot be predicted by measuring plasma protein levels but may be discoverable by genetic analysis.
Plasma Complement C4 Level
Dense Deposit Disease, C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome
Complement C4 (MW: 188 kDa), a central complement component in the classical and lectin pathways, is required to generate C4b2a, the C3 convertase of the classical pathway. C4 is cleaved to C4a (anaphylatoxin; 8 kDa) and C4b (180 kDa). C4b binds C2, and after removal of non-catalytic domain on C2, the C4b2a complex is formed. Unlike plasma C3, plasma C4 levels are typically normal in aHUS and C3G, however this test helps to rule out other complement-mediated renal diseases.
Soluble C5b-9 Assay
The complement system consists of three initiating pathways - the classical, alternative and lectin - activation of which leads to the generation of C3 and C5 convertases. The latter initiates the sequential activation of the terminal pathway (C5 to C9) resulting in the formation of pore-forming membrane attack complex C5b-9 (MAC), a stable complex that mediates irreversible cell lysis. Fluid-phase C5b-9 complexes (soluble C5b-9) may also form in complex with regulatory proteins like protein S.
C3 Glomerulopathy (C3G): The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. Soluble C5b-9 is elevated in both DDD and C3GN, but only the difference between C3GN and controls reaches statistical significance (p<0.001 for only C3GN) (see Zhang et al., Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).
Atypical Hemolytic Uremic Syndrome: Soluble C5b-9 levels are elevated in the majority of patients with active atypical hemolytic uremic syndrome but less frequently when the disease is in remission. Soluble C5b-9 may be useful as a biomarker to monitor activity of terminal pathway of complement (Bu et al., Soluble C5b-9 as a biomarker for complement activation in atypical hemolytic uremic syndrome, AJKD 2015).
Note: ADAMTS-13 Activity Assay is available 'a la carte'.
Comments:
Please print, complete and submit the Kidney Testing Requisition Form from the Molecular
Otolaryngology & Renal Research Laboratory, to Specimen
Control/Mailouts with the specimen and the Epic Requisition.
Methodology:
Enzyme Linked Immuno-Sorbent Assay (ELISA)
Modified Enzyme Linked Immuno-Sorbent Assay (ELISA)
Radial immunodiffusion (RID)
Modified Enzyme Linked Immuno-Sorbent Assay (ELISA)
Radial immunodiffusion (RID)
CPT Code:
FH Autoantibody-MORL - 83516
CH50eq-MORL - 86161
Alternative Pathway Functional Assay (APFA)-MORL - 86161
Hemolytic Assay-MORL - 86161
sMAC-MORL - 83520
C4 Level-MORL - 86160
C3 Level-MORL - 86160
Bb Fragment Level-MORL - 86160
FB Level-MORL - 86160
FH Level-MORL - 86160
FI Level-MORL - 86160
CH50eq-MORL - 86161
Alternative Pathway Functional Assay (APFA)-MORL - 86161
Hemolytic Assay-MORL - 86161
sMAC-MORL - 83520
C4 Level-MORL - 86160
C3 Level-MORL - 86160
Bb Fragment Level-MORL - 86160
FB Level-MORL - 86160
FH Level-MORL - 86160
FI Level-MORL - 86160