Cryohydrocytosis: When Cold Breaks the Membrane

An 8-week-old male with reported family history of spherocytosis (HS) was referred to hematology clinic for evaluation. He was born prematurely due to preterm labor after a 33-week gestation and experienced congenital non-immune hemolytic anemia and prolonged neonatal hyperbilirubinemia treated with phototherapy. Complete blood count (CBC) at 8 weeks of age demonstrated a hemoglobin of 8.8 g/dL, platelet count of 477 × 103/μL, and absolute reticulocyte count of 0.2526 × 106/μL, 9.8% of red blood cells (RBCs). Numerous stomatocytes were visible on peripheral blood smear (Figure 1A), prompting further investigation. Genetic evaluation with Next Generation Sequencing (NGS) of a red cell membrane disorders gene panel identified a heterozygous known pathogenic variant in SLC4A1 (NM_000342.4): c.2286T>A, p.(Ser762Arg). The resulting band 3 protein, which normally functions as the main RBC anion exchanger, is altered by this missense variant into a temperature-sensitive, leaky cation conduit causing cryohydrocytosis (CHC) [1, 2]. The father, who had been given a presumed diagnosis of HS since childhood, was found to have the same SLC4A1 variant. Osmotic gradient ektacytometry performed using the proband's and his father's blood, soon after the blood draw at room temperature (RT), demonstrated decreased Omin (the hypotonic osmolality at which 50% of the cells hemolyze in an osmotic fragility assay), decreased deformability as indicated by a decreased EImax (maximum Elongation Index), and a decreased Ohyp (the hypertonic osmolality where the Elongation Index is half of EImax), consistent with increased intracellular viscosity [3]. The pathognomonic finding of cryohydrocytosis emerged after overnight storage of the blood specimens at 4°C. Ektacytometry after 14 and 24 h at 4°C demonstrated a gradual rightward shift with a dramatic increase in Omin and Ohyp, reflecting marked osmotic changes of the patient's erythrocytes after cold exposure (Figure 1B). RBC histograms of the father's blood generated by an Advia Hematology Analyzer (Siemens, Malvern, PA) also demonstrated temperature changes. When maintained at RT and analyzed soon after collection, RBCs were normocytic with a mildly increased hemoglobin concentration (HC). After storage at 4°C for 24 h a significant decrease in the RBC HC and an increase in the RBC volume were observed (Figure 1C). Measurement of intracellular erythrocyte cation content confirmed abnormal membrane permeability for sodium (Na+) and potassium (K+) cations. Blood collected from the proband and father, maintained at RT and analyzed within 2 h contained RBCs with slightly elevated intracellular Na+ and decreased intracellular K+, reflecting a mild dehydration status at baseline. After cold storage for 24 h, the abnormal membrane permeability in cryohydrocytosis allowed for massive influx of Na+ along with K+ leak, causing overhydration and hemolysis (Figure 1D). CHC is a rare, autosomal dominant, hemolytic anemia classified as a subtype of stomatocytosis and caused by certain missense variants in SLC4A1, which cluster within transmembrane helices 8–12, the region of the protein normally involved in anion transport [4, 5]. RBCs demonstrate temperature-dependent cation transport abnormalities, with pronounced sodium influx and potassium efflux at 4°C–8°C, whereas at 37°C, the cation leak is mild (partially offset by an overactivated Na+/K+ pump), and it is minimal around 23°C (RT) [2, 5, 6]. A distinctive laboratory finding is pseudohyperkalemia due to potassium leak from fragile RBCs during specimen handling and cold storage before processing [7, 8]. Clinically, patients present with hemolytic anemia of mild or moderate severity, jaundice, fatigue, and splenomegaly. Symptoms and findings overlap with those of other forms of stomatocytosis, emphasizing the importance of RBC permeability studies and genetic testing for a definitive diagnosis. Patient education about cold avoidance and maintaining their specimens at RT prior to analysis is essential for accurate results. Management is primarily supportive. Splenectomy as a therapeutic approach is contraindicated because it does not improve the hemolysis and incurs a high risk of thromboembolic complications and pulmonary hypertension. Prophylactic anticoagulation should be considered in patients with CHC who have previously undergone splenectomy [9, 10]. The authors have nothing to report. This study was approved by the Institutional Review Board (IRB) of Cincinnati Children's Hospital Medical Center (IRB# 2011-1510). Informed consent was obtained from the family. The authors declare no conflicts of interest. All data and information concerning this study will be made available from the corresponding authors upon reasonable request.