What is the approach for testing complement levels in patients with suspected complement system dysfunction, such as Paroxysmal Nocturnal Hemoglobinuria (PNH) or atypical Hemolytic Uremic Syndrome (aHUS)?

Approach to Testing Complement Levels in Suspected Complement System Dysfunction

For patients with suspected complement system dysfunction such as Paroxysmal Nocturnal Hemoglobinuria (PNH) or atypical Hemolytic Uremic Syndrome (aHUS), a systematic approach to complement testing should begin with first-level laboratory tests including C3, C4, CH50, and AP50, followed by more specialized genetic and functional testing as indicated by initial results.

Initial Diagnostic Approach

• When suspecting complement-mediated disorders like aHUS, first assess for the clinical triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal involvement, though not all three signs may be present at disease onset 1

• Obtain haptoglobin, indirect bilirubin, and lactate dehydrogenase (LDH) levels as initial screening tests for any patient presenting with anemia plus thrombocytopenia to evaluate for possible hemolysis 1

• Measure complement C4 levels as the best initial screening test for hereditary angioedema (HAE), as all untreated patients with C1INH deficiency typically have low C4 levels 1

• When TMA (thrombotic microangiopathy) is suspected, urgently determine ADAMTS13 activity to distinguish between aHUS and TTP (thrombotic thrombocytopenic purpura) 1

First-Level Complement Tests

• First-level laboratory tests for complement disorders should include complete blood count, peripheral blood smear (for schistocytes), reticulocyte count, direct and indirect Coombs tests, LDH, haptoglobin, and renal function tests 1

• Measure C3 and C4 complement components as part of the initial evaluation for suspected complement disorders 1, 2

• Assess CH50 (classical pathway, 50% hemolytic activity) and AP50 (alternative pathway, 50% hemolytic activity) to evaluate overall complement function 1, 2

• Note that normal complement levels do not exclude complement-mediated disorders, as levels may be normal between attacks or early in disease 1, 2

Second-Level Complement Tests

• For suspected aHUS, proceed with second-level laboratory tests including genetic testing and immunologic diagnostic tests 1

• Perform genetic testing through next-generation sequencing of complement-related genes: CFH, CFHR1-5, C3, CD46, CFI, THBD, DGKE, and CFB 1

• Consider multiplex ligation-dependent probe amplification of genes CFH, CFHR1, CFHR2, CFHR3, CFHR4, and CFHR5 for suspected aHUS 1

• Include testing for anti-nuclear antibodies (ANA) as part of second-level laboratory tests for aHUS 1

Special Considerations for Different Disorders

For aHUS:

• Monitor parameters such as C3, C4, CH50, and AP50 when managing patients on C5 inhibitor therapy (eculizumab/ravulizumab), particularly if extending the interval between doses 1

• Complete suppression of complement activity is typically observed 1-3 weeks after eculizumab infusion, with partial suppression after 4 weeks 3

• Consider both genetic variants and autoantibodies when evaluating adults with aHUS, though gene variants appear more commonly than autoantibodies 1

For PNH:

• Evaluate for deficiency of complement regulatory proteins CD59 and CD55, which makes PNH cells susceptible to hemolysis 4

• Consider flow cytometry analysis of red blood cells and white blood cells to detect GPI-anchored protein deficiencies characteristic of PNH 4

For Hereditary Angioedema:

• Measure C1INH antigen and function if C4 level is low to distinguish between type I HAE (low C1INH antigen) and type II HAE (normal C1INH antigen but low function) 1

• Use a chromogenic functional C1INH assay for greatest sensitivity in detecting C1INH functional deficiency 1

• Measure C1q level (not C1q binding) to differentiate hereditary from acquired C1INH deficiency; C1q should be normal in HAE but decreased in acquired C1INH deficiency 1

Monitoring Complement During Treatment

• For patients receiving C5 inhibitors (eculizumab/ravulizumab), regular monitoring of complement activity can help optimize therapy and potentially allow for extended dosing intervals 3

• Complement functional tests (CH50, AP50) are useful for monitoring the efficacy of eculizumab treatment, with complete suppression indicating effective therapy 3

• Monitor disease activity markers (haptoglobin, LDH, platelet count) alongside complement tests to ensure disease remains in remission despite any adjustments to treatment intervals 3

Practical Challenges and Considerations

• Many specialized complement tests are not routinely available in commercial laboratories and may require sending samples to specialized centers 1

• Proper collection and storage of samples for complement testing is critical; consult with clinical laboratories about specific requirements 1

• Consider that Chinese and Japanese patients may have polymorphic variants of the C5 gene that can lead to resistance to anti-C5 monoclonal antibodies 1

• Genetic counseling should be provided to patients with confirmed complement-mediated disorders that have genetic components 1

• Remember that the absence of schistocytes should not exclude an early diagnosis of TMA due to the low sensitivity of this test 1