A reliable procedure for decontamination before thawing of human specimens cryostored in liquid nitrogen Three washes with sterile liquid nitrogen SLN2

Three subsequent washings in certified ultraviolet sterile liquid nitrogen produce an efficient decontamination of vitrification carriers, allowing safe thawing of human specimens.

Lodovico Parmegiani, M.Sc., Antonio Accorsi, M.Sc., Silvia Bernardi, B.Sc., Alessandra Arnone, B.Sc., Graciela Estela Cognigni, M.D., Marco Filicori, M.D.

Volume 98, Issue 4, Pages 870-875, October 2012


To report a washing procedure, to be performed as frozen specimens are taken out of cryobanks, to minimize the risk of hypothetical culture contamination during thawing.

Basic research.

Private assisted reproduction center.

Two batches of liquid nitrogen (LN2) were experimentally contaminated, one with bacteria (Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophilia) and the other with fungi (Aspergillus niger). Two hundred thirty-two of the most common human gamete/embryo vitrification carriers (Cryotop, Cryoleaf, Cryopette) were immersed in the contaminated LN2 (117 in the bacteria and 25 in the fungi-contaminated LN2). The carriers were tested microbiologically, one group without washing (control) and the other after three subsequent washings in certified ultraviolet sterile liquid nitrogen (SLN2). The carriers were randomly allocated to the “three-wash procedure” (three-wash group, 142 carriers) or “no-wash” (control group, 90 carriers) using a specific software tool.

Mean outcome measure(s):
Assessment of microorganism growth.

In the no-wash control group, 78.6% of the carriers were contaminated by the bacteria and 100% by the fungi. No carriers were found to be contaminated, either by bacteria or fungi, after the three-wash procedure.

The three-wash procedure with SLN2 produced an efficient decontamination of carriers in extreme experimental conditions. For this reason, this procedure could be routinely performed in IVF laboratories for safe thawing of human specimens which are cryostored in nonhermetical cryocontainers, particularly in the case of open or single straw-closed vitrification systems.

  • lodo

    Hi Nico. Sorry, I’ve seen only now your comment.

    (Do you know any method to link comments on this forum by email?)

    This additional handling in SLN2 requires only few minutes and it is not deleterious to the specimens (which remain frozen). so… why not? This pathogens have not caused crossed contamination but they could potentially affect the culture. Furthermore, this is a straightforward procedure to prove that you are doing anything possible to minimize any risk of culture contamination, and this can be reported on QC.

    I hope to see you soon



  • NicoGarrido

    Congratulations to the authors for their nice experimental work
    Nevertheless, as recognized in the manuscript, there is no report about cross contamination between human reproductive tissues or cells immersed in liquid or vapour nitrogen, nor reports about lower results in IVF as a consequence of this contamination, even with open systems.
    Moreover, subsequent sperm, oocyte and embryo washes after thawing may be enough to remove any infectious particle
    could the cleaning method itself be deletereous for the samples?
    the extent of gamete and embryo handling procedures is well known to affect viability
    Do the authors think it worths this additional handling the benefits obtained: removing pathogens that have not caused a crossed contamination in the clinical environment?

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