Anemia during Pegylated Interferon-Ribavirin Therapy Results from Both Increase Suppression of Erythroid Differentiation and Hemolysis
Authors: Ronzoni Luisa , Aghemo Alessio , Rumi Maria Grazia , Colancecco Alessandra , Monico Sara , Grillo Paolo , Colombo Massimo , Cappellini Maria Domenica 
Published: April 2020

Abstract
Anemia is a common side effect of Pegylated Interferon (PegIFN) and Ribavirin (RBV) combined therapy in patients with chronic hepatitis C (HCV). Hemolysis as consequence of RBV concentration in erythrocytes is the most accepted underlying mechanism; however, since the degree of treatment related anemia is highly variable, other pathogenic mechanisms could be hypothesized. The aim of this study was to evaluate the prevalence of hemolysis in patients treated with the combination of PegIFN and RBV compared to patients on monotherapy with PegIFN and to assess other possible mechanisms underlying anaemia. We studied 18 patients with chronic hepatitis C HCV-2 treated with PegIFN-α2a (180 mcg/week) plus RBV (800 mg/day) for 24 weeks and 10 patients with chronic hepatitis B treated with PegIFN-α2a 180 mcg/week monotherapy for 48 weeks. All the patients developed anemia after 24 weeks of treatment, although the mean hemoglobin decrease was higher in HCV patients (2.77 vs 1.82 g/dL at week 24, p=0.03), particularly at week 4 (1.86 vs 0.51 g/dL, p=0.007). Only 3 out of 18 HCV patients (16%) developed hemolytic anemia, documented by a marked increase in reticulocytes (3,4%) and in LDH levels (502 U/L) and by a significant decrease in haptoglobin levels (40,3 mg/dL). These patients showed a sharper and faster decrease of Hb compared to the remaining 15 HCV patients (week 4: 3.40 vs 1.55 g/dL, p=0.01). None of the HBV patients on treatment developed hemolytic anemia. To understand possible mechanisms underlying anemia, we evaluated ex vivo the effect of the therapy on erythropoiesis, through peripheral erythroid progenitors cell coltures and gene expression analysis during treatment. Peripheral blood mononuclear cells were plated in methylcellulose-supporting media at a concentration of 2×105 cells/mL and colony formation (BFUe and CFU-GEMM) was analyzed after 14 day of culture. Expression of γ-globin and GATA2 gene was evaluated with quantitative real-time PCR. HCV patients with hemolysis had an increase in BFUe number at week 4, whereas patients without signs of hemolysis showed a decrease in colonies formation at week 4 (baseline: 15.5 to 7.5 colony / 105 cells). A decrease in BFUe number was also observed in HBV patients along PegIFN treatment period. In all the HCV and HBV patients the reduction in BFUe number was associated with an increase in undifferentiated CFU-GEMM colonies (BFUe: 87 to 76% in HCV, 91 to 64% in HBV; CFU-GEMM: 13 to 24% in HCV, 9 to 36% in HBV). This suggested that the inhibitory effect of PegIFN on erythroid differentiation, supported also by increased expression of primitive erythropoiesis specific genes (γ-globin and GATA2 genes), plays a major role in causing anemia even in combined treatment with RBV. The hemolysis as cause of anemia so far attributed to RBV is apparently restricted to few cases and deserves further investigation.