Clinical Lab Tests
Specialty Diagnostic Testing
Tests Available
- ADAMTS13 Activity, Reflex to Inhibitor Assays
Overview
ADAMTS13 results will be reported before 5 pm on the day of sample receipt to enhance the ability to make the diagnosis of TTP quickly and allow more rapid and appropriate intervention with definitive therapy, specifically plasmapheresis.
ADAMTS13, a serum protease that cleaves multimers of von Willebrand Factor into smaller oligomeric fragments. ADAMTS13 activity is severely reduced in acquired and congenital forms of thrombotic thrombocytopenic purpura (TTP), leading to very large circulating multimers of von Willebrand Factor, and may be moderately reduced in other conditions such as disseminated intravascular coagulation, sepsis, hepatic dysfunction, and pregnancy. Assessment of ADAMTS13 activity is an important component of the initial assessment of a patient with evidence for a thrombotic microangiopathy, such as atypical hemolytic uremic syndrome (aHUS) and TTP, suggesting atypical hemolytic uremic syndrome if ADAMTS13 activity is normal or moderately reduced, and TTP if ADAMTS13 activity is severely reduced. ADAMTS13 testing will be offered in a reflex testing algorithm.
This panel testing for ADAMTS13 Activity utilizes a reflexive testing algorithm. 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%.
Test Descriptions
ADAMTS13 Activity
ADAMTS13 is a serum protease that cleaves ultralarge 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
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.
ADAMTS13 Inhibiting Antibody
Autoantibodies (typically IgG) that neutralize or enhance clearance of ADAMTS13 may severely inhibit ADAMTS13 activity and therefore allow the accumulation of ultralarge 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.
Tests Available
- Eculizumab Level
- Classical Pathway Function Assay (CH50)
Overview
Adequate dosage and dosing interval of eculizumab results 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.
The eculizumab level along with clinical markers of disease activity and other laboratory measures of terminal complement pathway activity such as CH50, and sC5b-9, should be used to guide therapeutic decision making as to adequate dosage and dosing interval.
Test Descriptions
Eculizumab Level
The eculizumab level performed by the Nephrology Clinical lab is an ELISA-based testing methodology that measures the free level of eculizumab in serum. 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).
Classical Pathway Function Assay (CH50)
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.
This test is useful to monitor patients with either atypical hemolytic uremic syndrome (aHUS) or other forms of thrombotic microangiopathy (TMA) being treated with eculizumab. 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.
Tests Available
- Classical Pathway Function Assay (CH50)
- Alternative Pathway Function Assay
- Lectin Pathway Function Assay
Overview
The complement system screen identifies abnormalities in the complement system by measuring each complement pathway. This analysis may be used to determine the cause of complement dysfunction and to monitor the effect of certain treatments. Complement levels may be decreased due to increased consumption or hereditary deficiency Decreased complement activity may be seen within a variety of conditions, including: Recurrent microbial infections (usually bacterial), autoimmune diseases (systemic lupus erythematosus and rheumatoid arthritis) and hereditary and acquired angioedema. Primary kidney diseases associated with low complement levels include membranoproliferative glomerulonephritis, post-infectious glomerulonephritis and atypical hemolytic uremic syndrome. Low complement levels are also present in cirrhosis, malnutrition and serum sickness.
Test Descriptions
Classical Pathway Function Assay (CH50)
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.
This test is useful to monitor patients with either atypical hemolytic uremic syndrome (aHUS) or other forms of thrombotic microangiopathy (TMA) being treated with eculizumab. 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.
Alternative Pathway Function Assay
The pathway is triggered when the C3b protein directly binds a microbe. It can also be triggered by foreign materials and damaged tissues . The Alternative Pathway acts as a powerful amplifier of both Classical Pathway and Lectin Pathway as the C3b formed by these pathways promote the formation of both C3 convertase and C5 convertase, thus forming an amplification loop.
Active C3b binds to the cell wall components and lipopolysaccharide and the constant low level of spontaneous C3b formation ensures that C3b can bind to invading cells and trigger the rest of the alternative complement pathway to lyse the cells.
The potential activation of the alternative pathway is kept in check by a natural inhibition, factor H and factor I. Factors H and I in plasma inactivate C3b enzyme in solution but C3b on cell surfaces cannot be inactivated due to protection by the bound properdin.
Lectin Pathway Function Assay
The Lectin and Classical pathway are identical except for the initiation products. Opsonin, a mannose-binding lectin (MBL) antibody and ficolins initiate the lectin pathway. Once MBL binds to mannose residues on the pathogen surface, activation occurs with serine proteases MASP1 and MASP2.MASP1 and MASP2 act as amplifiers in the MBL and ficolin complex in order to break apart C2 and C4. C4 cleaves into C4a and C4b and C2 into C2a and C2b.C4b and C2b forms C3 convertase (C4b2a).C3 convertase joins with C3b to make C5 convertase (C4b2aC3b).
Tests Available
- C3
- C4
- Factor B
- Bb
- Factor H
- Factor I
- C3 Nephritic Factor
- Factor H Autoantibody
Overview
Thrombotic microangiopathy (TMA) is a category of diseases linked by a common pathomechanism of endothelial injury leading to microvascular thrombosis and fibrin deposition, aggregation of platelets on the damaged endothelium, and organ dysfunction related to microvascular injury. This category includes Shiga toxin-producing E. coli associated hemolytic uremic syndrome (STEC-HUS), atypical hemolytic uremic syndrome (aHUS), and thrombotic thrombocytopenic purpura (TTP), among others. Atypical HUS is associated with defects in the regulation of the alternative complement pathway leading to uninhibited formation of the C3 convertase C3bBb on the endothelium and subsequent microvascular injury, whereas TTP is caused by the inability of ADAMTS13 to cleave ultra large multimers of von Willebrand Factor (vWF), with resulting adhesion of these ultra large vWF multimers to the endothelial surface under shear stress leading to microvascular thrombosis and injury. The evaluation of thrombotic microangiopathy includes testing the regulatory components of the alternative complement pathway for aHUS and the activity of ADAMTS13.
Please see Cincinnati Children's Hospital comprehensive platform of molecular and cellular diagnostic testing for thrombotic microangiopathies (TMA).
Test Descriptions
C3
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.
C4
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.
Factor B Level
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.
Bb
The complement Factor Bb level provides a quantitative assessment of the extent of activation of the alternative pathway of complement. 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.
Factor I Level
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.
Factor H Level
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.
C3 Nephritic Factor
An autoantibody that depresses the serum level of C3 and, secondarily, other complement components. Among the hypocomplementemic glomerulonephritides, nephritic factor is absent in acute post-streptococcal glomerulonephritis and lupus nephritis. It is present in most hypocomplementemic patients with membranoproliferative glomerulonephritis and also those with partial lipodystrophy. Although the antibody may be instrumental in producing the nephritis, its level is not closely associated with the clinical course.
Factor H Autoantibody
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.
Tests Available
- C1 inhibitor
- C1q
- C2
- C3
- C4
- C4 Binding Protein
- C5
- C6
- C7
- C8
- C9
- Properdin
Note: Tests can be performed individually, or in a profile package.
This profile is useful in the detection of inherited complement deficiencies that may predispose to lupus-like syndromes, to recurrent bacterial infection, particularly Neisserial, or to hereditary angioedema. In association with the measurement of nephritic factor, this profile aids in distinguishing acute from chronic nephritis and in identifying the three types of membranoproliferative glomerulonephritis.
Specimen requirements 1 ml serum, frozen.
Tests Available
- Cystatin C
Overview
Autoantibodies against M-type phospholipase A2 (PLA2R) receptors are highly specific for the diagnosis of primary, or idiopathic, membranous nephropathy (IMN) and can be detected in 70%-75% of patients with the disease. This test quantitates the level of anti-PLA2R in blood samples which facilitates a rapid diagnosis of IMN, helps to distinguish IMN from other glomerulonephritides, corresponds with the activity of the disease, and may be used to assess response to therapy. (Beck et al. NEJM. 2009)
Cystatin C measurements are used in the diagnosis and treatment of renal diseases.
It is a cysteine proteinase inhibitor and is formed by all nucleated cells. As it is formed at a constant rate and freely filtered by the healthy kidney it can be used for assessing renal function. Serum concentrations of Cystatin C are almost totally dependent on the glomerular filtration rate. A reduction in the GFR results in a rise in the concentration of Cystatin C. Cystatin C has not been shown to be affected by factors such as muscle mass and nutrition, factors which have been demonstrated to affect creatinine values. In addition, a rise in creatinine does not become evident until the GFR has fallen by approximately 50%.
Tests Available
- PLA2R Autoantibody
Overview
Autoantibodies against M-type phospholipase A2 (PLA2R) receptors are highly specific for the diagnosis of primary, or idiopathic, membranous nephropathy (IMN) and can be detected in 70%-75% of patients with the disease. This test quantitates the level of anti-PLA2R in blood samples which facilitates a rapid diagnosis of IMN, helps to distinguish IMN from other glomerulonephritides, corresponds with the activity of the disease, and may be used to assess response to therapy. (Beck et al. NEJM. 2009)
Tests Available
- C3, C4
- IgG, IgA, IgM
- Transferrin
- Albumin
- CRP
Overview
The SPP is useful in diagnosing dysgammaglobulinemias, iron deficiency, idiopathic nephrotic syndrome of childhood, active systemic lupus erythematosus and hypocomplementemic glomerulonephritides. The profile also aids in the assessment of the status of the immune system in patients receiving chemotherapy and may be abnormal in the presence of viral infections, the IgA nephropathies and acute and chronic inflammation.
Tests Available
- C1 inhibitor
- C2
- C3
- C4
This profile distinguishes HANE from other urticarias.
Specimen requirements 1 mL serum, frozen.
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Shipping
Please ship samples frozen, on dry ice, overnight to:
Cincinnati Children’s Hospital Medical Center
Nephrology Laboratory T6.325
240 Albert Sabin Way Dock S
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Contact:
To Obtain Lab Results (Professionals Only):
To obtain lab results, healthcare professionals can call 513-636-4281.
General Information:
The Nephrology Clinical laboratory may be reached at 513-636-4530.
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