Decreased 2,3 BPG level accounts for increased Hb oxygen affinity in Piezo1 HX. The 2,3-BPG level, expressed as 2,3-BPG/Hb ratio, was measured from fresh blood kept on ice and deproteinized using perchloric acid. (A) The 2,3-BPG/Hb ratio was significantly decreased in Piezo1 HX patients compared with controls (0.48 ± 0.09, n = 7 vs 1.01 ± 0.1, n = 4; P < .001). (B) The p50 values measured using an Hemox Analyzer showed that hemoglobin-oxygen affinity was higher in Piezo1-HX RBC compared with HS or controls RBC (Piezo1-HX: n = 11, p50 = 21.1 ± 0.9 mm Hg; HS: n = 4, p50 = 25.5 ± 0.5 mm Hg; controls: n = 10, p50 = 25.9 ± 0.7 mm Hg). The difference between Piezo1-HX and other conditions was statistically significant (P < .001). (C) p50 values measured using a venous blood gas analyzer confirmed the increased hemoglobin-oxygen affinity in Piezo1-HX RBC compared with Gardos-HX or HS-RBC (Piezo1-HX: n = 9, p50 = 22.2 ± 1.1 mm Hg; HS: n = 11, p50 = 26.4 ± 0.8 mm Hg; Gardos-HX: n = 2, p50 = 28.2 and 26.9 mm Hg, respectively; reference range, 25-29 mm Hg). One patient with Piezo1/Band3 double heterozygosity has a normal p50 (p50 = 25.4 mm Hg). The differences between Piezo1-HX and HS was statistically significant (P < .001). (D) Hemox Analyzer oxygen equilibrium curve showed a typical increase in Hb oxygen affinity for a Piezo1-HX RBC suspension (red line) compared with a normal control (dashed line) and Gardos-HX RBC (light red line).