Table 2.

Monitoring of eculizumab therapy and complement activity in atypical hemolytic uremic syndrome (aHUS)

Parameter	Interpretation	Remarks
Serum eculizumab level	Target is set at trough levels of 50–100 μg ml−1 to fully block complement	Reports differ regarding the measurement of only the free proportion of eculizumab [36] versus measurement of eculizumab both free and bound to C5 [12, 14]. Furthermore, eculizumab can bind a maximum of 2 C5 molecules per eculizumab molecule and is able to bind both C5 as C5b incorporated in C5b-C9 complex. Hence, measurement of eculizumab can comprise free (excess) eculizumab, eculizumab bound to 1 C5, eculizumab bound to 2 C5, or in combination with C5b-C9 complexes. [38]. The assay used in the trials to determine the trough levels measured both bound and free proportion. [16, 39]
Eculizumab-C5 complex	In contrast with serum eculizumab levels, one could also determine eculizumab bound to C5, hence only the bound proportion of eculizumab is determined.	It is known that eculizumab can also bind C5b-C9. Furthermore, one eculizumab molecule could bind respectively 1 or 2 C5 molecules, hence the remaining capacity is unknown [40]. Clinical use is unknown.
Total complement activity (CH50)	CH50 levels correlated nicely with eculizumab serum trough levels, and suppressed CH50 (< 10%) is reached with trough levels > 30–50 μg ml−1 [28, 36, 39, 41]	There are different assays to measure CH50. With this test, total complement activity (also known as CH50) is tested to determine the capacity of patient serum to lyse sheep or chicken erythrocytes coated with antibodies. In the case of a functional complement system, the CP will be activated, consequently leading to C5b-C9 deposition on the erythrocytes and consequently cause hemolysis. With the Wieslab test, CH50 can be measured with C5b-C9 formation, detected using a C9 neoantigen, as read-out [41]. Recently, Willrich et al. reported CH50 measurement during eculizumab treatment with liposome immunoassay with stable and reliable results [29].
Alternative pathway activity (AP50)	AP50 levels can be suppressed by eculizumab, however ongoing activation has been noted despite adequate eculizumab levels [41, 42].	There are different assays to measure AP50. Specific assessment of alternative pathway activation is possible with a hemolytic assay based on untreated rabbit erythrocytes (AP50). Puissant-Lubrano et al. compared both the hemolytic assays as used in all trials with the Wieslab ELISA in 16 patients treated with eculizumab and found conflicting results [41]. Residual activity of the AP was observed with the hemolytic assay, in the presence of (sufficient) eculizumab levels. In contrast with the Wieslab ELISA which showed complete blockade of the AP. Moreover, they assessed sensitivity of all assays and concluded that the Wieslab ELISA is less sensitive, hence the residual activity measured with the hemolytic assay is most accurate [41].
C3d	C3d is a breakdown product of C3, hence elevated C3d complement levels reflect activation at level of C3 is present.	C3d levels are elevated in acute phase of aHUS and decreased in the majority of patient with eculizumab therapy [42, 43].
C3	Can be both normal as decreased in aHUS patients during acute phase and remission	[44]
C5	Eculizumab binds to C5. Eculizumab trough levels correlate with C5 levels.	C5 levels fluctuate between and within patients due to among others disease activity [29].
C5a	C5a is released after cleavage of C5. In the case of eculizumab therapy, C5 cannot be cleaved, hence less C5a is present.	Interestingly, values of C5a do not decrease to zero, although no C5a should be present in light of sufficient eculizumab [42]. Furthermore, C5a has a very short half-life of approximately 1 min.
Ex vivo endothelial cell assay	By determining the C5b-C9 deposition after adding patient serum on activated endothelial cells, complement blockade could be assessed with good reproducibility	This assay has one major drawback since it is a highly specialized technique which cannot be easily performed in any laboratory [44]. Although Noris et al. advocates that persistent complement activation tested ex vivo (whereas CH50 remained low) is a reason to increase eculizumab dosage or decrease interval, Merril et al. showed different results using a ham test [44, 45].
Ham test	The Ham test is modified from the assay used to detect PNH. By acidifying the patient serum, AP is activated and results in erythrocyte lysis in PNH. In the modified Ham test, PNH-like cells are incubated with serum of aHUS patients and depending on AP dysregulation present in the serum, will be lysed [46].	Merril et al. showed no correlation was seen between positive or negative Ham test, hence the presence of complement activation and aHUS recurrence. Moreover, various patients remained positive with the Ham test without disease recurrence and withdrawal of eculizumab therapy [45].
Soluble C5b-C9 (TCC)	Soluble C5b-C9 should decrease during eculizumab therapy	Various studies report different results. Due to the ability of eculizumab to bind C5b-C9, it could be possible that these complexes have a lower clearance, hence are elevated during remission [29, 36, 41, 42, 44].

AP alternative complement pathway, CP classical complement pathway, ELISA enzyme-linked immunoabsorbent assay, PNH paroxysmal nocturnal hemoglobinuria, TCC terminal complement complex