The Effect of In Vitro Hemolysis on Measurement of Cell-Free DNA

Hemolysis during blood drawing is a common cause of laboratory artifacts. Although circulating cell-free tumor DNA and fetal DNA are currently measured in routine practice, the effect of in vitro hemolysis on the measurement of cell-free DNA (cfDNA) has not been investigated. When in vitro hemolysis occurs, cellular DNA could be released from damaged white blood cells and reduce the fraction of circulating tumor DNA and fetal DNA.Blood from healthy individuals was collected and passed through a narrow needle to cause in vitro hemolysis. Plasma was separated before and after mechanical damage, and concentrations of free hemoglobin and cfDNA of 2 reference genes were measured.cfDNA of 2 reference genes and free hemoglobin increased after mechanical damage. A clear correlation between cfDNA and free hemoglobin was observed.cfDNA concentrations are higher in hemolyzed plasma. Therefore, the fraction of circulating tumor DNA and fetal DNA can be underestimated in plasma hemolyzed by inappropriate blood collection techniques.