Serum Neutrophil Extracellular Trap Levels Predict Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation Yasuyuki Arai, Kouhei Yamashita,

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Serum Neutrophil Extracellular Trap Levels Predict Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation Yasuyuki Arai, Kouhei Yamashita, Kiyomi Mizugishi, Tomohiro Watanabe, Soichiro Sakamoto, Toshiyuki Kitano, Tadakazu Kondo, Hiroshi Kawabata, Norimitsu Kadowaki, Akifumi Takaori-Kondo Biology of Blood and Marrow Transplantation Volume 19, Issue 12, Pages 1683-1689 (December 2013) DOI: 10.1016/j.bbmt.2013.09.005 Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Survival of patients after allo-SCT. (A) Overall survival was calculated using the Kaplan-Meier method, and TMA and non-TMA groups were compared using the log-rank test. The 1-year overall survival was significantly lower (31.2% versus 69.8% [P < .01]) in the TMA group. (B) Nonrelapse mortality at 1- year was significantly higher (39.0% versus 9.3% [P = .03]) in the TMA group. Biology of Blood and Marrow Transplantation 2013 19, 1683-1689DOI: (10.1016/j.bbmt.2013.09.005) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Serum NET trends after allo-SCT. (A) The serum NET levels before conditioning regimen (PRE), on the day of SCT (Day0), and 4 weeks after SCT (4WK) in the TMA (N = 11) and non-TMA (N = 79) groups. (B) The ratio of serum NET levels at Day0 and 4WK to PRE (Day0/PRE and 4WK/PRE) in the TMA (N = 11) and non-TMA (N = 79) groups. In the TMA group, Day0/PRE and 4WK/PRE are significantly higher than in the non-TMA group. (C) The serum thrombomodulin levels before conditioning regimen (PRE), on the day of SCT (Day0), and 4 weeks after SCT (4WK) in the TMA (N = 11) and non-TMA (N = 34) groups. (D) The ratio of serum thrombomodulin levels at Day0 and 4WK to PRE (Day0/PRE and 4WK/PRE) in the TMA (N = 11) and non-TMA (N = 34) groups. *P < .05; **P < .01; ***P < .001. Biology of Blood and Marrow Transplantation 2013 19, 1683-1689DOI: (10.1016/j.bbmt.2013.09.005) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 High NET levels are a risk factor for TA-TMA. (A) The incidence of TA-TMA relative to the Day0/PRE serum NET ratios. (B) The incidence of TA-TMA relative to the 4WK/PRE serum NET ratios. (C) The incidence of TA-TMA relative to absolute serum NET levels at 4WK. Note that elevations of either the serum NET ratios (Day0/PRE and 4WK/PRE) or absolute NET levels at 4WK are significant risk factors for TA-TMA. Biology of Blood and Marrow Transplantation 2013 19, 1683-1689DOI: (10.1016/j.bbmt.2013.09.005) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Immunofluorescence staining of glomeruli in patients with TA-TMA. Kidney specimens were obtained at autopsy from patients with TA-TMA. (A) H & E staining; characteristic features of TA-TMA, such as fragmented erythrocytes (arrows), thrombosis, and fibrins (arrow heads) are visible. (B) Phosphotungstic acid hematoxylin staining and (C) periodic acid-Schiff staining showing fibrin deposition in glomeruli. (D and E) H & E staining of glomeruli in Cases 1 (D) and 2 (E). (F and G) Immunofluorescence staining with MPO and dsDNA in Cases 1 (F) and 2 (G). NETs are visible as weblike structures (arrow heads). The photographs in (F) and (G) are high-power views of the boxed areas in (D) and (E), respectively. Scale bars represent 50 μm (A-E) and 10 μm (F, G). Biology of Blood and Marrow Transplantation 2013 19, 1683-1689DOI: (10.1016/j.bbmt.2013.09.005) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions