Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Summary

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Abstract

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

Introduction

Multiple studies have characterized differences in gene expression, DNA methylation and cell subset in blood among individuals with and without mental (or other) illnesses^1-4. These studies, however, have been obtained from clinical settings in which disease-associated differences may be magnified due to the generally more severe nature of the illnesses for which patients are seeking treatment. Due to advances in “omics” approaches, the past decade has seen an explosion of interest in obtaining biologic samples from community and/or epidemiologic settings^5-7, in order to provide population-based estimates of disease prevalence and a broader pictu....

Protocol

The Detroit Neighborhood Health Study was reviewed and approved by the University of Michigan's Institutional Review Board. All participants provided informed consent prior to their participation in the study.

1. Overview

1. Properly document all stages from recruitment through analysis of endpoint data. Perform the protocols on Day 1 simultaneously, switching stages and overlapping as time permits. The sequence of stages is written to optimize performance efficiency. Figure 1 provides an overview of the entire process.
   Note: The term, “participant” is used throughout to signify the de-identi....

Results

It is essential that the overall procedure produce high quality material for analysis in a multitude of downstream applications including gene expression via microarray and RT-PCR analysis, detection of epigenetic modifications, and cell subset variations. Table 1 indicates the average yield and quality of materials from each of the processes. Figure 3 provides an example of the quality output of the leukocyte RNA isolation and filter processing methods. The image on the upper left of

Discussion

We have described a streamlined protocol that has been successfully applied to process more than 1,600 whole blood samples in the Detroit Neighborhood Health Study. Although many of these techniques are available in the existing literature, our step-by-step compilation, including precisely timed alterations between each step, reflects an optimized, efficient protocol that successfully produces a variety of biologic specimens with a wide range of downstream applications, including DNA methylation, mRNA expression and immu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
QIAamp DNA Blood Mini Kit	Qiagen	51104	Day 1: DNA isolation
Phosphate-buffered saline (PBS)	Sigma	P5493-1L	Day 1: PBMC isolation
5 ¾” Pasteur pipets	Fisher	13-678-6A	Day 1: PBMC isolation
Fetal Bovine Serum (FBS), heat inactivated	Life Technologies	10082147	Day 1: PBMC isolation
Dimethyl Sulfoxide (DMSO)	Sigma	D8418-500ml	Day 1: PBMC isolation
RPMI Medium 1640, liquid	Invitrogen	11875119	Day 1: PBMC isolation
0.4% trypan blue stain	Invitrogen	T10282	Day 1: PBMC isolation
Countess Cell Counting Chamber	Invitrogen	C10283	Day 1: PBMC isolation
Countess Automated Cell Counter or cell counting device such as a microscope and hemocytometer	Invitrogen	C10281	Day 1: PBMC isolation
LeukoLOCK Fractionation & Stabilization Kit	Ambion	1933	Day 1: Leukocyte RNA isolation
25 G x 5/8 in. needles	Becton Dickinson	305122	Day 1: Leukocyte RNA isolation
Syringes (5 ml)	Becton Dickinson	309646	Days 1 and 2: Leukocyte RNA isolation
Denaturing Lysis Solution	Ambion	8540G	Day 2: Leukocyte RNA isolation
5 M NaCl	Life Technologies	24740011	Day 2: Leukocyte RNA isolation
TRI Reagent	Ambion	9738	Day 2: Leukocyte RNA isolation
Bromo-3-chloro-propane (BCP)	Sigma	B-9673	Day 2: Leukocyte RNA isolation
spin cartridges	Ambion	10051G	Day 2: Leukocyte RNA isolation
0.1 mM EDTA	Ambion	9912	Day 2: Leukocyte RNA isolation
DNA-free Kit	Ambion	AM1960	Day 2: DNase treament
RNA 6000 Ladder	Agilent	5067-1529	Day 2: Bioanalyzer analysis
RNA 6000 Nano Series II Kit	Agilent	5067-1511	Day 2: Bioanalyzer analysis
RNaseZAP	Ambion	AM8782	Day 2: Bioanalyzer analysis
Ethanol >99%	Sigma	E7023-500ml
Isopropanol >99%	Sigma	I9516-500ml
Nuclease-free ultra pure water	Invitrogen	9938
Pipette tips (nuclease-free)	Eppendorf	22491253
Pipetter (serological)	Eppendorf	2223020-4
Pipetters (for volumes under 1 ml)	Eppendorf	3120000-054
Pipettes (serological)	Fisher	13-678-27E
Controlled rate freezing containers	Nalgene	5100-0001
Cryoboxes (to hold 2 ml and 5 ml cryovials and 1.5 ml microcentrifuge tubes)	Fisher	03-395-464
Test tube rack	Thermo Scientific	14-804-134
15 ml polypropylene tubes	Fisher	14-959-49D
1.5 ml and 0.65 microcentrifuge tubes	Fisher	07-200-534 and 07-200-185
2 ml and 5 ml cryovials	Fisher	10-500-26 and 10-269-88F
8 ml CPT vacutainer	BD Biosciences	362761	2 tubes
6 ml K2 EDTA vacutainer	BD Biosciences	367863	2 tubes
8.5 ml SST vacutainer	BD Biosciences	367988	1 tube
Vortexer	Fisher	2215365
Dry bath incubator with heating block for microcentrifuge tubes	Fisher	11-715-1250
Filtration/vacuum system for use within the cell culture hood	Fisher	01-257-87
Fixed-angle rotor for microcentrifuge tubes with aerosol-tight lid	Eppendorf	22637002
Refrigerated centrifuge with a swing-bucket rotor and aerosol-tight caps for 16 x 125 mm vacutainers and 15 ml polypropylene tubes	Eppendorf	22628157	2, one does not need to be refrigerated
Nanodrop 2000 (recommended for accuracy of small volumes) or other spectrophotometric device	Fisher	13-400-411
Agilent Bioanalyzer	Agilent Technologies	G2940CA
Liquid nitrogen tank	Thermo Scientific	11-676-56
Sharps container	Fisher	22-037-970
Biological waste container	Thermo Scientific	1223P52
Biosafety Level 2  certified cell culture hood	Thermo Scientific	13-261-315