Novel method for reduction of virus load in blood plasma by sonication

Aim of the present study is the evaluation of ultrasound as a physical method for virus inactivation in human plasma products prior to transfusion. Our study is focused on achieving a high level of virus inactivation simultaneously leaving blood products unaltered, measured by the level of degradation of coagulation factors, especially in third world countries where virus contamination of blood products poses a major problem. Virus inactivation plays an important role, especially in the light of newly discovered or unknown viruses, which cannot be safely excluded via prior testing.

Taking into account the necessary protection of the relevant coagulation activity for plasma, the basis for a sterile virus inactivation under shielding gas insufflation was developed for future practical use. Influence of frequency and power density in the range of soft and hard cavitation on the inactivation of transfusion-relevant model viruses for Hepatitis-(BVDV = bovine diarrhea virus), for Herpes-(SFV = Semliki Forest virus, PRV = pseudorabies virus) and Parvovirus B19 (PPV = porcine parvovirus) were examined. Coagulation activity was examined via standard time parameters to minimize reduction of functionality of coagulation proteins. A fragmentation of coagulation proteins via ultrasound was ruled out via gel electrophoresis. The resulting virus titer was examined using end point titration.

Through CO₂ shielding gas insufflation-to avoid radical emergence effects-the coagulation activity was less affected and the time window for virus inactivation substantially widened. In case of the non-lipidated model virus (AdV-luc = luciferase expressing adenoviral vector), the complete destruction of the virus capsid through hard cavitation was proven via scanning electron microscopy (SEM). This can be traced back to microjets and shockwaves occurring in hard cavitation. The degree of inactivation seems to depend on size and compactness of the type of viruses. Using our pre-tested and subsequently chosen process parameters with the exception of the small PPV, all model viruses were successfully inactivated and reduced by up to log 3 factor. For a broad clinical usage, protection of the coagulation activities may require further optimization.

Building upon the information gained, an optimum inactivation can be reached via raising of power density up to 1200 W and simultaneous lowering of frequency down to 27 kHz. In addition, the combination of the two physical methods UV treatment and ultrasound may yield optimum results without the need of substance removal after the procedure.