Depletion of hepatitis C virus from procured kidneys using pulsatile perfusion preservation.
The safety of the use of kidneys procured from a hepatitis C virus (HCV)-positive cadaver donors in transplantation has recently been the subject of controversy. One factor that is important in determining the transmission of the virus and/or viral liver disease is the total viral inoculum to which the renal allograft recipient is exposed as a result of the transplant. We have studied the effect of a standard pulsatile renal preservation procedure and variations of it on the number of viral copies in organs from HCV-positive donors. An HCV-RNA quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was utilized with a recombinant competitive inhibitor substrate added after cDNA synthesis with the PCR primers within the relatively non-polymorphic 5' untranslated region of the HCV genome. Additionally, strain specificity was found to be detectable using a modification of the technique of restriction fragment-length polymorphism (RFLP-PCR), so that virus from the organ donor could be specifically identified and quantified in the recipient. It was observed that standard preservation procedures using pulsatile perfusion were able to eliminate 75% of the virus from the organ in 20 hr. By modifying this procedure to include additional wash steps and a second pulsatile perfusion, greater than 99% of the virus could be eliminated from the kidney. In a related study, we used quantitative PCR to study requirements for filtration of the virus using HCV-positive serum. It was found that a high-flow-rate ultrafilter with a molecular weight cut-off (MWCO) of 300,000 daltons placed in series to the preservation apparatus was very efficient in eliminating the virus from perfusion solution in less than 2 hr. It can therefore be proposed that with the use of these molecular techniques, pulsatile perfusion coupled with additional viral depletion steps (dilution, and/or filtration) may allow the practical reduction of HCV transmission risk in recipient follow-up studies. The means are thereby presented for similar manipulation of other known or, as yet, unknown transmissible agents.
Zucker
K
K
Department of Surgery, University of Miami School of Medicine, Florida.
Cirocco
R
R
Roth
D
D
Olson
L
L
Burke
G W
GW
Nery
J
J
Esquenazi
V
V
Miller
J
J
D001483:Base Sequence; D002102:Cadaver; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000382:microbiology*; D008107:Liver Diseases / Q000097:blood; D016031:Liver Transplantation / Q000009:adverse effects; D008969:Molecular Sequence Data; D009926:Organ Preservation / Q000379:methods*; D016133:Polymerase Chain Reaction; D011673:Pulsatile Flow; D012367:RNA, Viral / Q000032:analysis; D014462:Ultrafiltration; D014774:Virulence
D012367:RNA, Viral