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Conventional assays of omega-3 long chain polyunsaturated fatty acid (n-3 LCPUFA) status in humans involve venous blood collection and expensive, multi-step processes that limit their usefulness as screening tools. This study aimed to develop a capillary dried blood spot (DBS) system capable of protecting n-3 LCPUFA from oxidation for up to 2 months at room temperature (20-25°C). We demonstrated that a DBS system comprising both an antioxidant and chelating agent on silica-gel coated paper prevented any significant change in the n-3 LCPUFA profile after 2 months. Our DBS assay was then tested in fifty subjects, and this demonstrated the presence of strong and significant correlations between the results obtained from the DBS system and those obtained from conventional measures for all fatty acids, in particular the n-3 LCPUFA EPA and DHA (r>0.96, P

Analysis of an extensive database of human platelet 5-HT2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. Bmax declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity KD during the half-life of Bmax. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg protein ml (at 0.2 nM of ligand). Above 0.1 mg protein ml , percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 mg ml , well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration.

Different ways of eluting IgG antibodies against Toxoplasma gondii from blood spots on PKU filter papers from newborns were examined, and the eluate was analysed by Enzyme Immunosorbent Assay (EIA). 10-mm diameter discs (method A), and 3-mm discs (method B) were eluted overnight, and 3-mm discs were eluted directly for 2 hours in the EIA wells (method C). Two groups of paired samples were tested: blood spots on the PKU card from the newborn, cord blood, and a venous blood sample from the mother collected shortly after delivery (Group 1, 191 samples); or PKU cards from the newborns and serum collected from the mother during the first trimester (Group 2, 426 samples). The best results were found for Group 1; method A, a sensitivity of 100% (63/63), and a specificity of 100% (128/128) (r = 0.934, P < 0.0001), and for Group 2; method C, a sensitivity of 100% (125/125), and a specificity of 100% (301/301) (r = 0.822, P < 0.0001). When method C, was used on 188 samples stored for 16-18 month at -20 degrees C, a sensitivity of 100% (62/62) and a specificity of 99.2% (125/126) (r = 0.913, P < 0.0001) were found. The PKU filter paper method is reliable and reproducible, and can be used in screening projects based on analysis of neonatal samples, and in epidemiological studies on toxoplasmosis in pregnant women.

The use of dried blood spots lends itself to widespread application in large field studies, especially in remote areas. We present experience gained during a perinatal HIV transmission study in southern Africa in which dried blood spot samples were used for polymerase chain reaction (PCR) tests. In this study, 15,810 filter paper cards with dried blood spots were collected. Infants were seen at age 6 and 12 weeks, and PCR was routinely done in duplicate on each sample. Of 186 negative controls (infants born to HIV-negative women), two (1.1%) had a single strongly reactive PCR result; the repeated duplicates were both negative. In contrast, all 24 known positive samples were strongly positive in both tests. Results were available from 1,976 duplicate tests on 1,235 infants born to HIV-infected women. Based on the PCR result on a later sample, the positive predictive value was 97.6% if both replicates were strongly positive (absorbance: 0.8 OD450 U), 100% when one of the replicates was strongly positive, and 27% when one or both replicates were weakly positive (but none strongly positive). When both replicates were negative, the negative predictive value wasor = 96.2%. Thus, when a single HIV PCR test has a strongly positive result, the infant is very likely to be infected. A positive PCR result after age 1 month was 98.9% accurate in predicting antibody positivity after 15 months. Suggestions for sample collection, storage, and PCR testing are provided.15,810 filter paper cards with dried blood spots were collected during a perinatal HIV transmission study in southern Africa to be subjected to polymerase chain reaction (PCR) testing. Infants were seen at ages 6 and 12 weeks, with PCR routinely done in duplicate on each sample. Of 186 infants born to HIV-negative mothers, two had a single strongly reactive PCR result, while the repeated duplicates were both negative. All 24 known positive samples were strongly positive in both tests. Results were available from 1976 duplicate tests on 1235 infants born to HIV-infected women. Based upon the PCR result of a later sample, the positive predictive value was 97.6% if both replicates were strongly positive, 100% when one of the replicates was strongly positive, and 27% when one or both replicates were weakly positive. When both replicates were negative, the negative predictive value was greater than or equal to 96.2%. Therefore when a single HIV PCR test has a strongly positive result, the infant is very likely infected. A positive PCR result after age 1 month was 98.9% accurate in predicting antibody positivity after 15 months.

The effect of storage on (1) amplifiability of nucleic acid (present at low level) and (2) properties of whole blood polymerase chain reaction (PCR) inhibitors (present at high levels) in Guthrie card bloodspots was evaluated. Natural PCR inhibitors (protein, hemoglobin, iron) were selectively eluted from Guthrie cards (1 to 30 mo storage) under nondenaturing conditions and quantitated. The PCR was performed by direct amplification. It was found that PCR inhibitors become increasingly resistant to elution ("fixed") over time. For example, 600 micrograms protein, 1.87 au hemoglobin, and 374 ng iron were solubilized from 1 mo bloodspots. In contrast, only 137 micrograms protein (22 percent), 0.34 au hemoglobin (18 percent), and 147 ng iron (39 percent) were solubilized from 30 mo bloodspots. Fixation does not result from excessive desiccation since bloodspot weight 2.20 mg +/- 0.21 (1 mo) and 1.92 mg +/- 0.31 (30 mo) was not significantly changed (p0.05). The majority of protein was characterized as albumin, and two rbc metal-containing proteins, carbonic anhydrase and hemoglobin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Despite the presence of "fixed" PCR inhibitors, it was found that bloodspots stored 1 to 30 mo could be amplified for two regions (98 bp and 491 bp amplicons) encoding the delta F508 cystic fibrosis mutation. It is suggested that nucleic acid also becomes "fixed" to the filter paper matrix and accounts, in part, for the ability to amplify Guthrie cards by direct PCR and low yield of deoxyribonucleic acid (DNA) reported for microextraction methods.

Blood specimens from 133 patients clinically diagnosed as dengue virus infection by physicians in Nakhon Phanom Hospital, Thailand, were examined to detect anti-dengue IgM and IgG antibodies by antibody capture ELISA. The blood specimens were divided into 3 types of storage; (1) frozen serum aliquots, (2) whole blood dried on filter paper strips, and (3) sera dried on filter paper strips. These specimens were stored for the periods of 1, 3, 4, and 5 months, at -20 degrees C in the case of frozen serum aliquots, or at room temperature in the case of specimens dried on filter paper strips, before examined in paralleled by the ELISA. Anti-dengue IgG antibodies were stable for at least 5 months of storage as dried whole blood or serum on filter paper strips. So were the anti-dengue IgM antibodies in the dried whole blood from secondary dengue cases. Anti-dengue IgM antibodies from primary dengue cases declined slowly in whole blood and more rapidly in serum, both dried on filter paper strips. In the serum dried on filter paper strips, even anti-dengue IgM antibodies from secondary cases decreased significantly on storage. We suggest that diagnosis on dengue infections by IgM-capture ELISA should be performed within 1 month after the test specimens are collected as whole blood, not as serum, when the filter paper method is used for sample collection.

Previous studies have shown that in sickle trait (AS) donor blood very little sickling is visible. AS donor blood was evaluated by a method of blood filtration, which would be sensitive to intracellular gellation of S hemoglobin which might not be evident morphologically. Blood was collected from normal and sickle trait subjects into CPD solution and stored at 4 C. Blood was removed over a 21-day period and filtered through paper filters at unit gravity and also examined microscopically. Sickling was not evident in saline wet preparations, but was seen in each AS sample in 10 per cent formalin-saline. At 4, 25, and 37 C, AS blood filtered less rapidly than did normal (P < 0.01). AS blood filterability became progressively prolonged as temperature rose; conversely normal blood filterability shortened with increasing temperature. Over 21 days, AS blood remained less filterable than normals. AS samples with high hemoglobin S percentages filtered significantly more slowly than did those with less S hemoglobin. Recognizing that these studies refer only to blood in vitro, no conclusions are drawn with respect to reversibility of the altered filterability or any possible clinical implications.

Posttransfusion purpura typically occurs in PLA1 negative blood recipients who have been previously immunized to the PLA1 antigen. Following transfusion, severe thrombocytopenia develops with the formation of anti-PLA1. Since the patients' platelets lack the PLA1 antigen, one would not expect this antibody to destroy autologous platelets. In this study we show that PLA1 antigen exists in stored blood and can absorb to PLA1 negative platelets making them PLA1 reactive. Incubating PLA1 (-) platelets with ultracentrifuged plasma from PLA1 (+) blood donors allowed anti-PLA1 to bind to PLA1 (-) platelets. Control plasma from PLA1 (-) blood donors did not lead to anti-PLA1 binding. Using an inhibition assay, we showed that stored blood contains PLA1 material that was not removed by ultracentrifugation. The material absorbing to PLA1 (-) platelets represented the PLA1 antigen, which was confirmed by Western blotting. After incubating plasma containing PLA1 antigen with PLA1 (-) platelets, reactivity at 95,000 D was observed. Native PLA1 (+) platelets showed a similar band. When PLA1 (-) platelets were incubated with plasma from a PLA1 (-) donor, this band was not present. These studies show that a soluble form of PLA1 antigen exists in stored blood that can absorb to PLA1 (-) platelets. Consequently, anti-PLA1 can bind to these platelets leading to thrombocytopenia. These observations may explain the autologous destruction of platelets in posttransfusion purpura.

Human sera were absorbed into electrophoretic paper (Whatman) discs. After drying and storage either at +4 °C or at room temperature (18 °C) for four, seven and 14 weeks, the sera were eluted with distilled water and the diphtheria antitoxin contents were determined. Comparison with the titration of sera stored in the conventional way showed that there had been a significant decrease in titre in the case of discs dried and stored at room temperature. Alternatively, when stored at +4 °C a significant decrease in titre did not take place. This method of storage can thus be used where deep freezing of serum is not available. The storage and the transportation of sera treated in this way is very convenient.

Serum and blood samples dried on filter paper discs and normal sera were collected from 142 Liberians with proven schistosomiasis and 25 Liberians without schistosomiasis. These samples were tested by the indirect hemagglutination test (IHA) employing lyophilized sheep erythrocytes sensitized with either cercarial or adult Schistosoma mansoni antigens. The following results were obtained: Out of the 142 sera collected from the schistosomiasis patients 130 (91.5%) reacted with the cercarial antigen and only 89 (62.6%) reacted with the adult S. mansoni antigen. None of the 25 sera from persons free of schistosomiasis reacted with either antigen. The sensitized lyophilized erythrocytes showed no loss of antibody activity after laboratory storage at 4 degrees C for 4 months which was interrupted twice for 16 hours each during shipment. Filter paper serum and blood samples were stored at -25 degrees C up to 84 days with 16 hours interruption during shipment. 77.6% of the eluates from blood showed the same antibody titer levels as in normal serum; 10% showed lower antibody titers and 11.5% had negative results compared with normal serum. Results obtained with eluates from dried serum were somewhat better. An additional storage of the dried blood specimens for ten days at room temperature resulted in a rise of the proportion of negative titers from 11.5% to 34.1%. This loss of antibody activity was found in two thirds of the samples with low antibody titers. The results indicate that blood and serum samples dried on filter paper platelets should be stored at low temperatures (-25 degrees C) to preserve the antibody response.

To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials.