Improving Access to Care: Enhancing Stability of Amino Acids Exposed to Heat and Humidity Using Pretreated Dried Blood Spot Cards
Biochemical/Metabolic and Therapeutics
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Introduction:
Amino acids are vital metabolites used to form proteins and enzymes, to act as neurotransmitters, act as energy sources, to affect epigenetic regulation, and to function as an intermediate in many critical metabolic pathways. Given their importance, many disorders result in perturbations in amino acid concentrations. Dried blood spots (DBSs) provide an expedient method for the collection of multiple time points from a single specimen for research as well as diagnosis and monitoring of patients with inborn errors of metabolism. DBSs are an attractive sample modality for two reasons: 1) the ease and low cost of sample collection, as it can be obtained at home and without need for medical supervision, and 2) simple sample storage and transport. Amino acids as a DBS sample, however, are susceptible to environmental conditions such as high heat and humidity which can be encountered during storage and transport of the sample. We sought to improve the stability of amino acids in a DBS when exposed to harsh conditions to improve sample integrity and to improve the measurement quality of these analytes.
Methods:
Guthrie filter paper cards (Whatman 903 paper) were prepared by soaking the entire sample collection area in different pretreatment solutions (Phosphoric acid, Acetic acid/White vinegar, EDTA, or CVS Health ethylenediaminetetraacetic acid (EDTA) containing over the counter eye drops). The cards were allowed to fully dry and for the dual treatment conditions soaked a second time with the corresponding solution (either EDTA then Phosphoric acid or Phosphoric acid then EDTA). DBSs were prepared using 75µL aliquots from a single fresh sample of whole blood, collected into a tube without additives or preservatives. The blood spots were allowed to dry for a minimum of 4 hours then either: immediately analyzed, per our previously described HPLC methodology, or separated to the various exposure conditions for 7 full days before analysis. Exposure conditions for the DBS were as a measure of barrier protection (unprotected, hard plastic as a 50mL sealed conical tube with desiccant, or biohazard bag with desiccant) and environmental conditions (room temperature with no control of humidity, 37˚C without high humidity, and 37˚C with humidity greater than 92%).
Results:
Concentrations greater than 3N of acid led to increased fragility of the card and initial assessments showed no improvement when comparing 3N to 1N acid at room temperature conditions. For potential “at home” use, the lower normality of acid was used for the remaining experiments. At home approved pretreatment solutions (over the counter EDTA containing eye drops and food grade acetic acid) were also pursued but both were found to underperform the untreated card at room temperature conditions. Under high heat and humidity, pretreatment of the card and the use of a barrier significantly increased the consistency of sample recovery, compared to untreated with or without a barrier. Sample precision was also significantly improved by the presence of a barrier for multiple pretreatment conditions. EDTA pretreatment with a barrier, compared to untreated with a barrier, additionally improved overall recovery and accuracy at 7 days of high heat and humidity, with an additional significant increase in the number of analytes that were within 20% of the baseline measurement, which was whole blood analyzed directly after collection. EDTA pretreatment also significantly decreased the average percent change in recovery between Day 0 and Day 7.
Conclusion:
Pretreatment of filter paper cards used for the collection of DBS samples with EDTA allows for a significant improvement in the recovery and stability of amino acids exposed to high heat and humidity, even for 7 continuous days, and allows for greater precision and accuracy of sample analysis.
Amino acids are vital metabolites used to form proteins and enzymes, to act as neurotransmitters, act as energy sources, to affect epigenetic regulation, and to function as an intermediate in many critical metabolic pathways. Given their importance, many disorders result in perturbations in amino acid concentrations. Dried blood spots (DBSs) provide an expedient method for the collection of multiple time points from a single specimen for research as well as diagnosis and monitoring of patients with inborn errors of metabolism. DBSs are an attractive sample modality for two reasons: 1) the ease and low cost of sample collection, as it can be obtained at home and without need for medical supervision, and 2) simple sample storage and transport. Amino acids as a DBS sample, however, are susceptible to environmental conditions such as high heat and humidity which can be encountered during storage and transport of the sample. We sought to improve the stability of amino acids in a DBS when exposed to harsh conditions to improve sample integrity and to improve the measurement quality of these analytes.
Methods:
Guthrie filter paper cards (Whatman 903 paper) were prepared by soaking the entire sample collection area in different pretreatment solutions (Phosphoric acid, Acetic acid/White vinegar, EDTA, or CVS Health ethylenediaminetetraacetic acid (EDTA) containing over the counter eye drops). The cards were allowed to fully dry and for the dual treatment conditions soaked a second time with the corresponding solution (either EDTA then Phosphoric acid or Phosphoric acid then EDTA). DBSs were prepared using 75µL aliquots from a single fresh sample of whole blood, collected into a tube without additives or preservatives. The blood spots were allowed to dry for a minimum of 4 hours then either: immediately analyzed, per our previously described HPLC methodology, or separated to the various exposure conditions for 7 full days before analysis. Exposure conditions for the DBS were as a measure of barrier protection (unprotected, hard plastic as a 50mL sealed conical tube with desiccant, or biohazard bag with desiccant) and environmental conditions (room temperature with no control of humidity, 37˚C without high humidity, and 37˚C with humidity greater than 92%).
Results:
Concentrations greater than 3N of acid led to increased fragility of the card and initial assessments showed no improvement when comparing 3N to 1N acid at room temperature conditions. For potential “at home” use, the lower normality of acid was used for the remaining experiments. At home approved pretreatment solutions (over the counter EDTA containing eye drops and food grade acetic acid) were also pursued but both were found to underperform the untreated card at room temperature conditions. Under high heat and humidity, pretreatment of the card and the use of a barrier significantly increased the consistency of sample recovery, compared to untreated with or without a barrier. Sample precision was also significantly improved by the presence of a barrier for multiple pretreatment conditions. EDTA pretreatment with a barrier, compared to untreated with a barrier, additionally improved overall recovery and accuracy at 7 days of high heat and humidity, with an additional significant increase in the number of analytes that were within 20% of the baseline measurement, which was whole blood analyzed directly after collection. EDTA pretreatment also significantly decreased the average percent change in recovery between Day 0 and Day 7.
Conclusion:
Pretreatment of filter paper cards used for the collection of DBS samples with EDTA allows for a significant improvement in the recovery and stability of amino acids exposed to high heat and humidity, even for 7 continuous days, and allows for greater precision and accuracy of sample analysis.