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Berzuini et al report the observation that nearly half of patients with COVID-19 tested at their blood center had a positive direct antiglobulin test (DAT). However, eluates did not react with any test cells but did react with red cells from other patients with COVID-19 that were DAT negative. This suggests that COVID-19 may modulate the red cell membrane and present novel antigenic epitopes.

The antiviral drug ribavirin (RBV) is widely used in combination with pegylated interferon (PegIFN) for the treatment of hepatitis C virus (HCV) infection. A major side effect of RBV is a reversible anemia. The mechanism underlying anemia in RBV treated patients remains speculative although hemolysis was suggested. To get further insights, we evaluated in vitro the effect of RBV and PegIFN on proliferation and erythroid differentiation of CD34 pheripheral blood cells. The mononuclear cells derived from pheripheral blood of healthy volunteers were enriched for CD34+ cells by positive selection using anti-CD34-tagged magnetic beads. CD34+ cells were cultured for 14 days with a specific medium containing erythropoietin to induce erythroid differentiation. RBV (0.5mM and 0.1mM) and PegIFN (100U and 1000U) were added alone or in combination at different days of culture: precisely at day 0 corresponding to CD34 stage and at day 7 corresponding to proerythroblast stage. Cells growth and viability were evaluated by tripan blue exclusion; erythroid differentiation was investigated by cytofluorimetric analysis of Glicophorin A (GPA) expression, by morphological analysis on benzidine-May-Grunwald-Giemsa stained smears and by erythroid specific (γ-globin and GATA2) gene expression analysis with real-time PCR. RBV added at day 0 of culture drastically inhibited cell growth and differentiation (RBV 0.1mM: 8.5-fold reduction in cells growth and 1.6-fold reduction in GPA+ cells vs untreated cells at day 14; RBV 0.5mM: 12-fold reduction in cells growth and 30-fold reduction in GPA+ cells vs untreated cells at day 14). Addition of RBV at day 7 of culture had a milder inhibitory effect (RBV 0.1mM: 2.4-fold reduction in cells growth and 0.7-fold reduction in GPA+ cells vs untreated cells at day 14; RBV 0.5mM: 3.6-fold reduction in cells growth and 2.3-fold reduction in GPA+ cells vs untreated cells at day 14). Addition of PegIFN both at day 0 and day 7 of culture delayed erythroid differentiation, with reduction of GPA+ cells, decrease of orthocromatic erythroblasts and increase of more undifferentiated polychromatophilic erythroblasts, and increased expression of primitive erythropoietic genes (tab 1). The combined addition of PegIFN and RBV both at day 0 and day 7 of culture affected cell growth and erythroid differentiation less than RBV alone, with increase of GPA+ cells, decrease of basophilic erythroblasts and increase of more differentiated orthocromatic erythroblasts, and decreased expression of γ-globin gene. However cells treated with the combination of PegIFN and RBV were more undifferentiated than cells treated with PegIFN alone. All these observations confirmed the inhibitory effect of PegIFN on erythroid differentiation and strongly suggested an inhibitory action of RBV on cell growth, particularly on undifferentiated stem cells. These observations provide a model for the pathophysiology of anemia observed with ribavirin treatment. Tab. 1 % GPA+ cells % polychromatophilic e. % orthocromatic e. γ-gene GATA2 gene Untreated cells 61 17 55 1 1 PegIFN 100U 50 37 56 1,75 1,16 PegIFN 1000U 47 57 23 2,39 1,7

Chronic neutropenia (CN) is defined as a blood absolute neutrophil count (NC) below 1500/ml, lasting for at least 6 months. We report the results of serological screening for anti-neutrophil antibodies, performed in our laboratory during 2002–2005, in 100 consecutive children affected from CN and followed in 4 Italian pediatric centers. The patients, 65 males (m) and 35 females (f), had a median age of 18 months (range: 1–180). Both IgG bound to patient neutrophils (CD16+ cells) and the patient serum reactivity against healthy donor granulocytes (Direct and Indirect Granulocyte-Immunofluorescence Test, D-GIFT and I-GIFT) were assessed by flow-cytometry. The results are shown in the table, grouped according to the pattern of reactivity observed with D- and I-GIFT: A NC above 1500/ μl on the day of serological screening was found in 5.5%, 22.4%, 0% and 35% of patients in groups 1, 2, 3 and 4, respectively. Secondary CN, usually in association with other autoimmune disorders or malignant diseases, was reported in 5.5%, 10.3%, 0% and 50% of patients in groups 1, 2, 3 and 4, respectively. Infections were present in 72%, 79%, 25% and 8% of patients in groups 1, 2, 3 and 4, respectively. In group 2, 50 of the 58 patients (86%), who didn’t show anti-neutrophil antibodies in their sera, presented neutropenia with concomitant infections. In these patients only D-GIFT was able to detect neutrophil antibodies. Patients testing positive for I-GIFT had more severe neutropenia. Patients negative by both techniques were mostly found to have non-immunomediated neutropenia at clinical follow up. Although many laboratories perform only an indirect test to detect antineutrophil antibodies, our results suggest that direct test is more sensitive in identifying immunomediated chronic neutropenia. table group GIFT no. of patients m/f NC (cells/μl)* WBC (x102/μl)* * Mean ± SD 1 D+ I+ 18 15/3 757 ± 520 7.58 ± 3.1 2 D+ I− 58 36/22 968 ± 818 7.29 ± 5.3 3 D– I+ 4 3/1 525 ± 369 6.71 ± 2.3 4 D- I− 20 11/9 1693 ± 1510 8.28 ± 3.2