Abstract
Intravascular hemolysis accompanies diverse diseases, yet blood-based markers (eg, plasma-free hemoglobin [PFH], haptoglobin, lactate dehydrogenase [LDH], and bilirubin) are variably sensitive, nonspecific, or impractical for point-of-care use. We evaluated urinary carbonic anhydrase 1 (CA1) as a mechanistically grounded, urine-based marker of hemolysis in a multicenter study spanning the United Kingdom, Bangladesh, and Peru. We enrolled 234 participants: healthy adults and adults with inherited anemias (Oxford), newborns in intensive care (London), children and adults with complicated or uncomplicated malaria (Bangladesh), and adults attending rural clinics for various medical reasons (Peru). Urine CA1 and hemoglobin (Hb) were quantified by enzyme-linked immunosorbent assay and immunoblot, with CA1:Hb stoichiometry used to distinguish intravascular hemolysis from urogenital blood contamination. Relationships with PFH, LDH, bilirubin, Hb, C-reactive protein (CRP), and clinical variables were tested using regression, principal component analysis, and decision tree. Reference urine samples were predominantly CA1-negative/Hb-negative. In inherited anemias, urinary CA1 was highest in sickle cell disease and correlated with serum LDH and inversely with blood Hb. In neonates, longitudinal CA1 trajectories stratified infants into physiological, transient, and sustained hemolysis groups; higher CA1 levels were associated with prematurity, elevated CRP and lower white blood cell count. Urinary CA1 was detected in Bangladeshi patients with elevated PFH and in cases of intravascular hemolysis not identified by PFH. Among Peruvian participants, urinary CA1 correlated with raised CRP level, consistent with inflammation being a prohemolytic trigger. To enable rapid and cost-effective testing, a lateral flow device was developed and verified for excellent sensitivity and specificity. Urinary CA1 provides a sensitive and practical readout of intravascular hemolysis suitable for point-of-care testing globally.