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Dried blood spots as a source of anti-malarial antibodies for epidemiological studies.

Corran PH, Cook J, Lynch C, Leendertse H, Manjurano A, Griffin J, Cox J, Abeku T, Bousema T, Ghani AC, Drakeley C, Riley E - Malar. J. (2008)

Bottom Line: 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.When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK. patrick.corran@lshtm.ac.uk

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 degrees 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.

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Efficiency of recovery of antibodies from blood spots. Comparison of OD obtained from paired plasma and blood spot eluates (each at an equivalent of 1:000 dilution) where blood spots were stored for 2 days (a,c) or 2 weeks at ambient temperature and RH (b,d). Samples were assayed for antibodies to MSP-119 (a,b) and MSP-2 (c,d). Aliquots of 8 hyperimmune plasma samples were mixed with equal volumes of erythrocytes, spotted on 3 MM paper and dried overnight. The lines in a,c are the best fit least square straight lines, calculated recovery 99%. The dashed lines in c and d represent the least-squares best fit lines for the recoveries shown.
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Figure 3: Efficiency of recovery of antibodies from blood spots. Comparison of OD obtained from paired plasma and blood spot eluates (each at an equivalent of 1:000 dilution) where blood spots were stored for 2 days (a,c) or 2 weeks at ambient temperature and RH (b,d). Samples were assayed for antibodies to MSP-119 (a,b) and MSP-2 (c,d). Aliquots of 8 hyperimmune plasma samples were mixed with equal volumes of erythrocytes, spotted on 3 MM paper and dried overnight. The lines in a,c are the best fit least square straight lines, calculated recovery 99%. The dashed lines in c and d represent the least-squares best fit lines for the recoveries shown.

Mentions: Eight hyperimmune African plasma samples were mixed with an equal volume of fresh erythrocytes and duplicate 10 μl aliquots were spotted onto 3 MM paper and dried. After 48 h at ambient temperature and RH, 2.5 mm discs were cut and incubated overnight at ambient temperature to elute the antibodies. The eluates were then assayed for total IgG to MSP-119 and MSP-2 and, in one experiment, for MSP-119 specific IgG1 and MSP-2-specific IgG3. The ODs obtained for the eluates were compared with those obtained for the corresponding plasma samples tested at the equivalent dilution. For both antigens the correlation between the OD values obtained for plasma and blood spot eluates was >0.98, with a recovery of >95% for total IgG (Figures 3a and 3c) and for IgG1 and IgG3. However, when blood spots were kept at ambient temperature and RH for two weeks, antibody recovery was much reduced, to approximately 40% of original concentration for total IgG for MSP-119 and 35% for MSP-2 (Figures 3b and 3d). This variability in recovery prompted us to investigate the rate at which antibodies decayed under different storage conditions.


Dried blood spots as a source of anti-malarial antibodies for epidemiological studies.

Corran PH, Cook J, Lynch C, Leendertse H, Manjurano A, Griffin J, Cox J, Abeku T, Bousema T, Ghani AC, Drakeley C, Riley E - Malar. J. (2008)

Efficiency of recovery of antibodies from blood spots. Comparison of OD obtained from paired plasma and blood spot eluates (each at an equivalent of 1:000 dilution) where blood spots were stored for 2 days (a,c) or 2 weeks at ambient temperature and RH (b,d). Samples were assayed for antibodies to MSP-119 (a,b) and MSP-2 (c,d). Aliquots of 8 hyperimmune plasma samples were mixed with equal volumes of erythrocytes, spotted on 3 MM paper and dried overnight. The lines in a,c are the best fit least square straight lines, calculated recovery 99%. The dashed lines in c and d represent the least-squares best fit lines for the recoveries shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567984&req=5

Figure 3: Efficiency of recovery of antibodies from blood spots. Comparison of OD obtained from paired plasma and blood spot eluates (each at an equivalent of 1:000 dilution) where blood spots were stored for 2 days (a,c) or 2 weeks at ambient temperature and RH (b,d). Samples were assayed for antibodies to MSP-119 (a,b) and MSP-2 (c,d). Aliquots of 8 hyperimmune plasma samples were mixed with equal volumes of erythrocytes, spotted on 3 MM paper and dried overnight. The lines in a,c are the best fit least square straight lines, calculated recovery 99%. The dashed lines in c and d represent the least-squares best fit lines for the recoveries shown.
Mentions: Eight hyperimmune African plasma samples were mixed with an equal volume of fresh erythrocytes and duplicate 10 μl aliquots were spotted onto 3 MM paper and dried. After 48 h at ambient temperature and RH, 2.5 mm discs were cut and incubated overnight at ambient temperature to elute the antibodies. The eluates were then assayed for total IgG to MSP-119 and MSP-2 and, in one experiment, for MSP-119 specific IgG1 and MSP-2-specific IgG3. The ODs obtained for the eluates were compared with those obtained for the corresponding plasma samples tested at the equivalent dilution. For both antigens the correlation between the OD values obtained for plasma and blood spot eluates was >0.98, with a recovery of >95% for total IgG (Figures 3a and 3c) and for IgG1 and IgG3. However, when blood spots were kept at ambient temperature and RH for two weeks, antibody recovery was much reduced, to approximately 40% of original concentration for total IgG for MSP-119 and 35% for MSP-2 (Figures 3b and 3d). This variability in recovery prompted us to investigate the rate at which antibodies decayed under different storage conditions.

Bottom Line: 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.When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK. patrick.corran@lshtm.ac.uk

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 degrees 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.

Show MeSH
Related in: MedlinePlus