Performing Assays in Whole Dried Blood Spots

Dried blood spots (DBS) have several benefits over commonly used sample matrices for example plasma, serum, and even liquid whole blood.

The key advantages are convenient sample collection, shipping, and storage.1,2 DBS can be gathered and delivered without the need for cold storage or freezing, taking away the need for freezers, a centrifuge, or even a phlebotomist.1

DBS ultimately allows for decentralized sample collection in applications whether in areas without the infrastructure of a laboratory, longevity studies which find patient retention difficult, or in the monitoring of chronic diseases.3,4

This article outlines how the Simoa Neuro 4-Plex Assay can analyze levels of three neuronal biomarkers, NF-Light (NfL), Tau, and GFAP, in normal, healthy dried blood spots.

Materials and Methods

Materials and Sample Processing

Liquid human whole blood was taken from four individuals into EDTA collection tubes. The dried blood spots were assembled utilizing 50 µL of whole blood per spot onto a Whatman® protein saver card.

The blood was spotted within five minutes of gathering and was then air-dried at room temperature. The control blood spots were stored frozen at −80 °C. All samples were measured employing the Simoa multiplex Neurology 4-Plex “A” (N4PA) assay. The samples were then analyzed and processed with the use of the HD-1 Analyzer.

Methods

The stability of the dried spots was evaluated by leaving the spots at room temperature for the particular number of days and then storing them at −80 °C.

Before testing with the N4PA assay, all spots were thawed at room temperature for one hour. The DBS was eluted in 500 µL N4PA kit sample diluent for four hours at room temperature on an orbital shaker at 500 RPM, and run neat on the HD-1 Analyzer.

Specificity was measured by immuno-depleting DBS samples of analyte. The eluted DBS samples were incubated while rotating at room temperature (RT), with a 10x number of assay kit capture beads for one hour.

Stability testing was carried out with the spots left at room temperature for either 0, 1, 3, 6, or 8 days to evaluate signal changes with time spent at RT.

Conclusion and Discussion

The N4PA kit demonstrates the feasibility to test Tau, NfL, and GFAP in dried whole blood. All three analytes can be measured above LOQ in normal dried whole blood. Tau signal diminishes by 72% to values higher than the calibrator blank. Both the NfL and GFAP signal depletes by 100% to values less than the calibrator blank.

The Tau signal remains stable for eight days at room temperature, with signals varying from 96.8% to 129.2% of Day 0. The NfL signal shows instability at room temperature. In the first day at room temperature, the signal increases between 131.2% to 355.6%.

The use of DBS with NfL is possible if the spots are placed in cold storage within one day of collection. It is interesting that this elevated signal depletes to 100% which indicates that it is likely specific.

It is thought that the degradation of associated proteins at room temperature may create an increased availability of NfL epitopes. Although, the reasons for this elevated signal have not been fully discovered. The GFAP signal remains stable for eight days at room temperature in three of the four samples tested, with signals varying from 95.1% to 114.0% of Day 0.

The N4PA kit does not present the ability to correctly measure UCH-L1 in dried whole blood. The signal is non-specific as samples measure above LOQ but the signal does not deplete.

References and Further Reading

  1. Carlson, B.F., et al. “Dried Blood Spots: An Evaluation of Utility in the Field.” Annals of Global Health, vol. 82, no. 3, 2016, p. 450., doi: 10.1016/j.aogh.2016.04.241.
  2. Evans, Christopher, et al. “Implementing Dried Blood Spot Sampling for Clinical Pharmacokinetic Determinations: Considerations from the IQ Consortium Microsampling Working Group.” The AAPS Journal, vol. 17, no. 2, 2014, pp. 292– 300., doi:10.1208/s12248-014-9695-3.
  3. Lim, Mark D. “Dried Blood Spots for Global Health Diagnostics and Surveillance: Opportunities and Challenges.” The American Journal of Tropical Medicine and Hygiene, vol. 99, no. 2, 2018, pp. 256–265., doi:10.4269/ajtmh.17-0889.
  4. Smit, Pieter W., et al. “Systematic Review of the Use of Dried Blood Spots for Monitoring HIV Viral Load and for Early Infant Diagnosis.” PLoS ONE, vol. 9, no. 3, 2014, doi:10.1371/journal. pone.0086461.

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Last updated: Aug 13, 2019 at 4:19 AM

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