Dried Blood Spots as a Source of Anti-malarial Antibodies for Epi Studies
Dried Blood Spots as a Source of Anti-malarial Antibodies for Epi Studies
Background: Blood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions.
Methods: Blood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda.
Results: Antibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4°C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values.
Conclusion: This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided.
When carrying out serological surveys, particularly in remote locations, it is of great advantage to have a method of collecting and storing blood samples which does not require that facilities for centrifugation are accessible, and which is relatively robust to irregular degrees of refrigeration, at least for short periods. One approach that seems to offer these advantages is to collect samples as dried blood spots, and to recover antibodies from the dried spots once transferred back to the laboratory. Blood spots have the advantage that they are quick, simple and inexpensive to prepare and to store, require very small blood volumes which can be obtained by finger- or heel-prick, and are likely to be more socially acceptable in cultural contexts in which larger volumes of blood are difficult to collect. Blood spots have been used routinely since the 1960s for neonatal screening, initially for phenylketonuria, but subsequently for many other biochemical assays, including the assay of specific enzymes, determination of metabolites by mass spectrometry and, on occasion, for measuring antibody levels. More recently blood spots have been used as a source of DNA for screening for genetic abnormalities in newborns, for example for cystic fibrosis and haemoglobinopathies. Blood spots have been used for monitoring antibodies against several viral, bacterial and other pathogens, storage of monoclonal antibodies and, increasingly, for screening for HIV infection, both as a source of antibodies and for virus detection by PCR. Dried blood spots have been particularly useful for isolating parasite DNA in mapping the spread of drug resistance in malaria parasites
Although the stability of low molecular weight analytes in blood spots has been extensively studied and guidelines have been produced for blood spot collection, transport and storage together with recommendations and structures for quality control and quality assurance, the recovery of antibodies from dried blood spots has been less thoroughly investigated. Although small-scale studies of antibody stability in blood spots have been reported, these studies were not designed to be of predictive use in assessing storage conditions. In preparation for use of blood spots to derive serological measures of malaria transmission intensity, a thorough analysis of the stability of anti-malarial antibodies in blood spots has been undertaken, based on the well-established techniques used for determining the stability of biological reference materials. This paper presents the results of these studies together with a validated protocol for collection, storage and use of blood spots for antibody quantitation.
Abstract and Background
Abstract
Background: Blood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions.
Methods: Blood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda.
Results: Antibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4°C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values.
Conclusion: This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided.
Background
When carrying out serological surveys, particularly in remote locations, it is of great advantage to have a method of collecting and storing blood samples which does not require that facilities for centrifugation are accessible, and which is relatively robust to irregular degrees of refrigeration, at least for short periods. One approach that seems to offer these advantages is to collect samples as dried blood spots, and to recover antibodies from the dried spots once transferred back to the laboratory. Blood spots have the advantage that they are quick, simple and inexpensive to prepare and to store, require very small blood volumes which can be obtained by finger- or heel-prick, and are likely to be more socially acceptable in cultural contexts in which larger volumes of blood are difficult to collect. Blood spots have been used routinely since the 1960s for neonatal screening, initially for phenylketonuria, but subsequently for many other biochemical assays, including the assay of specific enzymes, determination of metabolites by mass spectrometry and, on occasion, for measuring antibody levels. More recently blood spots have been used as a source of DNA for screening for genetic abnormalities in newborns, for example for cystic fibrosis and haemoglobinopathies. Blood spots have been used for monitoring antibodies against several viral, bacterial and other pathogens, storage of monoclonal antibodies and, increasingly, for screening for HIV infection, both as a source of antibodies and for virus detection by PCR. Dried blood spots have been particularly useful for isolating parasite DNA in mapping the spread of drug resistance in malaria parasites
Although the stability of low molecular weight analytes in blood spots has been extensively studied and guidelines have been produced for blood spot collection, transport and storage together with recommendations and structures for quality control and quality assurance, the recovery of antibodies from dried blood spots has been less thoroughly investigated. Although small-scale studies of antibody stability in blood spots have been reported, these studies were not designed to be of predictive use in assessing storage conditions. In preparation for use of blood spots to derive serological measures of malaria transmission intensity, a thorough analysis of the stability of anti-malarial antibodies in blood spots has been undertaken, based on the well-established techniques used for determining the stability of biological reference materials. This paper presents the results of these studies together with a validated protocol for collection, storage and use of blood spots for antibody quantitation.
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