VIROLOGIC ANALYSIS ON BIOLOGICAL SAMPLES COLLECTED AND STORED ON "FILTER PAPER"

2. ABSTRACT

VIROLOGIC ANALYSIS ON BIOLOGICAL SAMPLES COLLECTED AND STORED ON "FILTER PAPER"

Nitrocellulose filter paper has been used for the collection, storage and transport of some biological samples for various laboratory analysis.

This method was used for the first time in Scotland by Dr. Robert Guthrie in 1963 for the collection of blood samples. The sample is applied directly onto the filter paper and dried at room temperature or in a oven for hours. The protein components bond to RNA or DNA are stabilized on the matrix of the filter paper without the need of refrigerated environment which is necessary when routine biological samples cannot be processed within 6 hours from collection. Samples are stored in low gas permeability plastic bags, to reduce moisture and therefore to keep them at room temperature in geographical areas with tropical climate, even for a long period of time.

Samples on filter paper have been used for the screening of metabolic diseases, such as Phenylketonuria (PKU) and Sickle Cell Disease, and to assess retrospectively, in samples collected at birth, the presence of Viral infection in the newborn.

The purpose of this study is to evaluate detectability and Viral load of DNA Viruses in biological samples of various type collected and stored on filter paper. In particular, it was examined whether the viral load detected with routine methods remained unvaried after application and storage on filter paper.

96 clinical samples (peripheral blood, serum, bronchial alveolar wash and urine), routine

analyzed for the detection of DNA Viruses, at the Laboratory Unit of Virology, University

of Pisa, Azienda Universitario-Ospedariera Pisana, found positive for HBV (Hepatitis B

Virus), EBV (Epstein Barr Virus, HHV4) or CMV (Cytomegalovirus, HHV5) were placed

on paper "Whatman FTA cards" (Whatman). Following elution of the sample from filter paper, viral genomes were analyzed using the same method used for routine diagnosis.

Data obtained show that Viral nucleic acids are detectable on clinical samples spotted on filter paper. We observed a log reduction on sensitivity in the detection of EBV in bronco- alveolar wash and blood and in the CMV detection in blood samples.

HBV was detected continuously at all viral load examined with an average sensitivity within a logarithm.

The ease and low cost of this sampling technique, the small volume required (50μl) and the

stability of dried nucleic acids, suggest filter paper as a good method of collection, storage

and transport of, besides blood and sera, also bronco-alveolar wash for the detection of DNA

Viruses analyzed, especially in developing countries where refrigeration systems are not

available.