br Materials and methods Eculizumab levels were measured at

Materials and methods Eculizumab levels were measured at Cambridge Biomedical using a manual ELISA method in which the microtitre plates are coated with C5, then blocked. Standards, controls, and unknowns were added. After incubation and washing, mouse anti-human IgG4 horseradish peroxidase (HRP) was added as the conjugate. Then the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added and the color reaction was halted using a stop reagent. The results were read spectrophotometrically at 450nm using a plate reader [2]. The pharmacodynamics of eculizumab were determined by measuring the capacity of the patient׳s serum to lyse chicken leukotriene b4 in a standard total human serum-complement hemolytic assay [3].
Discussion sThe median age at diagnosis of PNH is 34 years, so affected females are often in the reproductive age group. PNH is not known to adversely affect female fertility. There are 6 reported cases of successful pregnancies in PNH patients being treated with eculizumab [4–7]. Kelly et al. described the first 3 women who received eculizumab during pregnancy [4]. Two of these patients suffered from breakthrough hemolysis in the third trimester. Breakthrough hemolysis manifested itself with patients experiencing hemoglobinuria prior to their next scheduled dose of eculizumab. Both of these patients were treated by reducing the interval between subsequent doses from 14 days to 12 days in one case and to 7 days in the other case. The third patient delivered at 28 weeks due to preeclampsia. Three other cases have been described [5–7]. One of them suffered breakthrough hemolysis and was treated by reducing the duration between eculizumab doses to 7 days [7]. We describe the seventh case of successful pregnancy in the era of eculizumab. Our patient was stable on standard dose eculizumab until the second trimester. It has been noted that the dose of eculizumab required to block complement during the latter stages of pregnancy is greater than in the non-pregnant state [4]. Our patient developed breakthrough hemolysis in the second trimester despite standard eculizumab administration. Her eculizumab level was sub-therapeutic and a pharmacodynamic assay demonstrated the absence of complement blockade. Rather than reducing the time interval between doses, we opted to escalate the dose of eculizumab. Higher doses of eculizumab have been needed to control atypical hemolytic uremic syndrome (aHUS) [8] and catastrophic antiphospholipid syndrome [9]. In aHUS, the recommended dose of eculizumab is 33% higher than for PNH in order to completely block the complement cascade [10]. Utilizing this escalated dose of eculizumab, we successfully achieved therapeutic eculizumab levels with adequate complement blockade (Fig. 2) and controlled the patient׳s hemolysis (Fig. 1). This dose of eculizumab was also recently reported in a pregnant patient with aHUS and found to be safe [11]. Pregnancy activates the complement cascade, with increased terminal complex formation in the third trimester [12]. We hypothesize that loss of control of complement-mediated hemolysis with eculizumab during the second and third trimester appears to be due to the physiologic expansion of intravascular volume which occurs during pregnancy. Our patient׳s serum eculizumab level was clearly subtherapeutic, reflecting the dilution which had occurred. Dose escalation restored the level to therapeutic range and controlled hemolysis. Eculizumab levels were not detectable in our patient׳s cord blood nor breast milk. It therefore does not appear to cross the placenta nor be excreted in breast milk, which is consistent with previous reports [4–7]. Forty-five percent of pregnancies in women with PNH result in spontaneous miscarriage and 6% in maternal mortality due to fatal thrombotic events [13]. Because of the risk of life threatening thrombotic complications, pregnant women with PNH should receive full anticoagulation during pregnancy and for at least 4–6 weeks postpartum [14].