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HelpOrder Code AHUSD Atypical Hemolytic Uremic Syndrome Complement Panel, Serum and Plasma
Useful For
Detecting deficiencies in the alternative pathway that can cause atypical-hemolytic uremic syndrome, dense deposit disease, and C3 glomerulonephritis
A second-tier test that aids in the differential diagnosis of thrombotic microangiopathies
Profile Information
| Test ID | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| INTGA | AHUS Interpretation | No | Yes |
| COM3 | Complement, Total, S | Yes, (order COM) | Yes |
| AH503 | Alternative Complement Path Func, S | Yes, (order AH50) | Yes |
| C3HUS | Complement C3, S | Yes, (order C3) | Yes |
| C4HUS | Complement C4, S | Yes, (order C4) | Yes |
| FBCA | Factor B Complement Antigen, S | No | Yes |
| FHCA | Factor H Complement Antigen, S | No | Yes |
| CBB | CBb Complement, P | No | Yes |
| SC5B9 | SC5b-9 Complement, P | Yes, (C5B9) | Yes |
Reflex Tests
| Test ID | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| C1Q | Complement C1q, S | Yes | No |
| C1QFX | C1Q Complement, Functional, S | Yes | No |
| C2FXN | C2 Complement, Functional, S, NR | Yes | No |
| C3FX | C3 Complement, Functional, S | Yes | No |
| C4FX | C4 Complement, Functional, S | Yes | No |
| C5FX | C5 Complement, Functional, S | Yes | No |
| C6FX | C6 Complement, Functional, S | Yes | No |
| C7FX | C7 Complement, Functional, S | Yes | No |
| C8FX | C8 Complement, Functional, S | Yes | No |
| C9FX | C9 Complement, Functional, S | Yes | No |
| C5AG2 | C5 Complement, Antigen, S | Yes, (order C5AG) | No |
Method Name
C3HUS, C4HUS, FBCA, FHCA; C5AG2: Nephelometry
COM3: Automated Liposome Lysis Assay
AH503, CBB, SC5B9: Enzyme-Linked Immunosorbent Assay (ELISA)
INTGA: Medical Interpretation
Reporting Name
aHUS Complement Panel, S and PSpecimen Type
Plasma EDTASerum
Ordering Guidance
This test should be performed prior to treatment initiation and in the absence of therapy with complement inhibitors, such as eculizumab or ravulizumab. Complement inhibitors will affect performance of these assays.
For evaluating patients with possible thrombotic microangiopathies (TMA), the recommended first-tier test is ADM13 / ADAMTS13 Activity and Inhibitor Profile, Plasma. This test should be a second-tier test for TMA.
For patients who have received eculizumab or need to monitor response to eculizumab therapy, the recommended test is ECMP / Eculizumab Monitoring Panel, Serum. Soluble membrane attack complex should not be used as a standalone assay to monitor eculizumab efficiency.
For patients who have received ravulizumab or need to monitor response to ravulizumab therapy, the recommended test is RAVMP / Ravulizumab Monitoring Panel, Serum. Soluble membrane attack complex (sMAC) should not be used as a standalone assay to monitor ravulizumab efficiency.
Specimen Required
Both serum and plasma are required for this test.
Patient Preparation:
1. Fasting preferred but not required.
2. Do not collect specimens for at least 48 hours following plasma exchange.
Supplies: Sarstedt Aliquot Tube, 5 mL (T914)
Specimen Type: Serum
Collection Container/Tube:
Preferred: Serum gel
Acceptable: Red top
Submission Container/Tube: Plastic vial
Specimen Volume: 1.5 mL total in 3 separate plastic vials, each containing 0.5 mL
Collection Instructions:
1. Immediately after specimen collection, place the tube on wet ice and allow specimen to clot.
2. Centrifuge at 4° C.
3. Aliquot serum into 3 separate plastic vials, each containing 0.5 mL.
4. Within 30 minutes of centrifugation, freeze specimen. Specimen must be placed on dry ice if not frozen immediately.
Note: If a refrigerated centrifuge is not available, it is acceptable to use a room temperature centrifuge, provided the sample is kept on ice before centrifugation, and immediately afterward, the serum is aliquoted and frozen.
Specimen Type: Plasma
Collection Container/Tube:
Preferred: Lavender top (K2 EDTA)
Acceptable: Lavender top (K3 EDTA), light-blue top (Sodium citrate)
Submission Container/Tube: Plastic vial
Specimen Volume: 1.5 mL total in 2 separate plastic vials, each containing 0.75 mL
Collection Instructions:
1. Immediately after specimen collection, place the tube on wet ice.
2. Centrifuge between 1000 and 2000 x g for 10 minutes at 4° C.
3. Aliquot plasma into 2 separate plastic vials, each containing 0.75 mL.
4. Within 30 minutes of centrifugation, freeze specimen. Specimen must be placed on dry ice if not frozen immediately.
Note: If a refrigerated centrifuge is not available, it is acceptable to use a room temperature centrifuge, provided the sample is kept on ice before centrifugation, and immediately afterward, the plasma is aliquoted and frozen.
Specimen Minimum Volume
Serum: 1.5 mL; plasma: 1.5 mL
Specimen Stability Information
| Specimen Type | Temperature | Time |
|---|---|---|
| Plasma EDTA | Frozen | 14 days |
| Serum | Frozen | 14 days |
Reject Due To
| Gross hemolysis | OK |
| Gross lipemia | Reject |
| Gross icterus | OK |
Reference Values
FACTOR B COMPLEMENT ANTIGEN
15.2-42.3 mg/dL
SC5b-9 COMPLEMENT
≤250 ng/mL
FACTOR H COMPLEMENT ANTIGEN
18.5 to 40.8 mg/dL
CBb COMPLEMENT ACTIVATION FRAGMENT
≤1.6 mcg/mL
COMPLEMENT C4
14-40 mg/dL
COMPLEMENT C3
75-175 mg/dL
ALTERNATIVE COMPLEMENT, PATHWAY (AH50) FUNCTIONAL
≥46% Normal
COMPLEMENT, TOTAL
30-75 U/mL
Method Description
Complement, Total:
An automated method is performed using liposomes as the target for the serum complement system. The dinitrophenyl (DNP)-labeled liposomes are sensitized with antibody to DNP. Serum complement causes lysis and release of entrapped glucose-6-phosphate dehydrogenase. Glucose-6-phosphate dehydrogenase reacts with glucose-6-phosphate and nicotinamide adenine dinucleotide (NAD[+]). NAD(+) is reduced to NADH and the conversion is measured at 340 nm. The assay correlates with the CH50 assay based on sheep red blood cell lysis, has lower variability, and is simpler to perform.(Package insert: Fujifilm Autokit CH50. Fujifilm Wako Pure Chemical Corporation; 04/01/2018; Yamamoto S, Kubotsu K, Kida M, et al. Automated homogeneous liposome-based assay system for total complement activity. Clin Chem. 1995;41[4]:586-590)
Alternative Complement, Pathway Functional:
The Wieslab enzyme-linked immunosorbent assay complement assay for the alternative pathway combines principles of the hemolytic assay for complement activation with the use of labeled antibodies specific for neoantigens produced as a result of complement activation. The micro titer plate strips are coated with lipopolysaccharide. Patient serum is diluted in diluent containing specific blocker to ensure that only the alternative pathway is activated. During the first incubation, the diluted patient serum in the wells is activated by the coating. The wells are then washed and C5b-9 (membrane attack complex: MAC) is detected with a specific alkaline phosphatase labeled antibody to the neoantigen expressed during MAC formation. After a final wash, an alkaline phosphatase substrate is added. The amount of alternative pathway complement activity correlates with the color intensity of the solution and is measured in terms of absorbance (optical density).(Frazer-Abel A, Sepiashvili L, Mbughuni MM, Willrich MA. Overview of laboratory testing and clinical presentations of complement deficiencies and dysregulation. Adv Clin Chem. 2016;77:1-75. doi:10.1016/bs.acc.2016.06.001)
Complements C3 and C4; C5, Factor B, and Factor H Complement Antigens:
In these Siemens Nephelometer II methods, the light scattered onto the antigen-antibody complexes is measured. The intensity of the measured scattered light is proportional to the amount of antigen-antibody complexes in the sample under certain conditions. If the antibody volume is kept constant, the signal behaves proportionally to the antigen volume.
A reference curve is generated by a standard with a known antigen content on which the scattered light signals of the samples can be evaluated and calculated as an antigen concentration. Antigen-antibody complexes are formed when a sample containing antigen and the corresponding antiserum are put into a cuvette. A light beam is generated with a light emitting diode, which is transmitted through the cuvette. The light is scattered onto the immuno-complexes that are present. Antigen and antibody are mixed in the initial measurement, but no complex is formed yet. An antigen-antibody complex is formed in the final measurement.
The result is calculated by subtracting value of the final measurement from the initial measurement. The distribution of intensity of the scattered light depends on the ratio of the particle size of the antigen-antibody complexes to the radiated wavelength.(Instruction manual: Nephelometer II Operations. Siemens, Inc; Version 2.3, 2008; Addendum to the Instruction Manual 2.3, 08/2017)
CBb Complement Activation Fragment:
Microtiter plates are coated with monoclonal antibody specific to the complement factor Bb (CBb) fragment of the fourth component of the complement cascade. Controls, standards, and patient samples are exposed to the plate. After washing the plate, a horseradish peroxidase-conjugated polyclonal CBb antibody is added followed by a substrate to initiate color change.(Package insert: MicroVue CBb Plus EIA Kit. Quidel Corporation; PIA027044EN00 07/2022)
SC5b-9 Complement Activation Complex:
Microtiter plates are coated with monoclonal antibody specific to the C9 ring of the soluble C5b-9 (sC5b-9) complex. Controls, standards, and patient samples are exposed to the plate. After washing the plate, a horseradish peroxidase-conjugated anti-sC5b-9 complex antibody is added followed by a substrate to initiate color change.(Package insert: MicroVue SC5b-9 Plus EIA Kit. Quidel Corporation; PIA020004EN00, 06/2022)
Day(s) Performed
Varies
Performing Laboratory
Mayo Clinic Laboratories in Rochester
CPT Code Information
86160 x 6
86161
86162
Forms
If not ordering electronically, complete, print, and send 1 of the following forms with the specimen:
-Renal Diagnostics Test Request (T830)
-Coagulation Test Request (T753)