The Filter Paper Leishmania impression (FPLI), normally stored in ehtanol is to be used for PCR later.
For cutaneous leishmaniasis, to collect a sample from a leison non-invasively, to do a PCR, FPLI are used. These filter paper (cut strips or punches) are then kept in ethanol, but I will be in a very remote field location and would want to know what is the maximum time this can be stored for, to be still able to use it for PCR, without jeopardizing the results.
Dear Rehman,
In our lab (Dermatomycology - University of Brasília) we have biopsy materials stored in filter papers for as long as 5 years in room temperature (25 - 30 celsius degrees) and the DNA is still ok most of the time. We believe that the dehydration caused by the filter paper can preserve the sample, although some DNA degradation is expected. We also don't use etanol as it can make your DNA extraction less precise. We store it in envelopes.
Make sure you will scrap a good quantity of cells in your filter paper. You should put your filter paper in contact with the edges of the leishmania lesion and make Strong movements to collect a good sample. The patient will refer a mild discomfort and discreet bleeding is possible. Dry and old lesions are difficult to be managed.
After, you let the sample dry and store it in an envolope (ideally plastic). Make sure samples don't touch each other to avoid contamination.
Prefer high sensitive extraqction Kits rather than simple slution. We use Nucleospin Tissue XS - MN.
Best Regards
Thanks a lot Gomes for your detailed answer. This is really helpful.
just to confirm, you take the impressions on the filter paper, let it dry and keep it in a plastic envelop at room tempratures??
One more question, do you have a publication in which you have stated this method??
Best,
KR
Dear Khalid Rehman,
Ideally, after collecting filter papers and let it dry you should extract the DNA immediately or store it in a -80 C refrigerator. When we create a protocol for research we try to follow this methods.
However, in your case you have a technical problem. You must collect samples in remote areas and than transport it. This is very important, because leishmaniasis is a neglected disease that appears in remote areas.
In diagnostic exams, we frequently need to transport (sometimes patients live in the Amazon Rain Forest) to remote areas, so we have no choice. In our lab experience we can store it at room temperature up to 5 years. We never measured the rate of DNA degradation during this time, but we have a significant conservation of DNA. The same is not true for RNA.
We have forensic techniques that use this method. Search for protocols of forensic filter papers.
See the article bellow (free full text). I suggest you to compare forms of storing your sample. It is a method to be studied.
In the attached file they stored the filter paper in 70% ethanol. This is another easy way to store and transnport it....
Am J Trop Med Hyg. 2001 Dec;65(6):902-6.
Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.
Marques MJ1, Volpini AC, Genaro O, Mayrink W, Romanha AJ.
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