Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Summary

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Abstract

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 °C or -80 °C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

Introduction

The main aim of the research into cancer biomarkers is the identification of new biological parameters that can be used for diagnosis, for predicting patient prognosis, and for determining whether a patient will respond to a specific treatment. This research area is fundamental for the discovery of innovative cancer treatments and plays a key role in tailored treatment.

The procedures carried out during each step of biomarker identification and validation must be reliable and reproducible. A cornerstone for the success of translational research is the correct storage of biological samples such as blood and serum. This is the first step towa....

Protocol

Peripheral blood and serum were collected and stored from healthy donors and patients who gave written informed consent to take part in the study. The study protocol was approved by the local Ethics Committee in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

1. Blood Storage in DBS

1. Blood sample collection
   1. Collect 3 mL of peripheral blood sample in a 3 mL tube with 5.4 mg of ethylenediaminetetraacetic acid (EDTA).
      NOTE: Store peripheral blood as DBS as soon as possible and within 8 h.
   2. Write the patient code number on the bottom right-hand corner....

Results

We took advantage of the DBS and DSS procedures to store blood and serum at room temperature without affecting the quality of the biological material. Figure 1 shows an example of the protein card saver without blood and after blood collection. In order to confirm that the storage on filter paper and the procedure to elute blood do not interfere with sample quality, we performed a comparison with standard storage methods. We extracted DNA from 3 matched sampl.......

Discussion

This protocol investigates the potential for storing blood and serum on filter paper when the labs do not have the staff or infrastructures needed for the correct handling of blood samples. In particular, whole blood or serum collected in standard tubes or by finger-prick can be stored using this method and there is no need to freeze samples at -20 °C or -80 °C immediately after the blood collection. DBS/DSS can remain at room temperature for up to 14 days without any change in blood/serum integrity. A critical.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Whatman protein saver cards 903 Protein saver card, 100/pk‎	Sigma	Z761575	Useful to store samples at room temperature for downstream analyses
Falcon Serological Pipettes, 5 mL	Stem cell	#38003
50-200 µL tips	Star-Lab	S1120-8810
1.5 mL centrifuge tube	Eppendorf	4036-3204
QIAamp DNA microkit	Qiagen	56304	This is a DNA extraction kit designed to isolate small quantities of DNA
Buffer ATL (included in QIAamp DNA microkit)	Qiagen		This is reported as Lysis Buffer 1 in the text
Buffer AL (included in QIAamp DNA microkit)	Qiagen		This is reported as Lysis Buffer 2 in the text
QIAamp mini elute column	Qiagen		This is reported as column in the text
AW1  (included in QIAamp DNA microkit)	Qiagen		This is reported as Wash Buffer 1 IN THE TEXT
AW2  (included in QIAamp DNA microkit)	Qiagen		This is reported as Wash Buffer 2 in the text
Human Vitamin D BP Quantikine ELISA Kit	R&D systems	DVDBP0