Table 2 Preanalytical factors for methylation analysis of the included studies

de Caceres [18]

Serum

1.5

Retrospective

N/A

N/A

Proteinase K/SDS at 37 °C overnight

Phenol/chloroform

Sodium bisulfite conversion

Su [19]

Serum

1

N/A

N/A

3000×g, 10 min

QIAamp DNA Blood Mini Kit (QIAGEN)

EZ DNA Methylation Kit (Zymo Research)

Modified DNA stored at − 80 °C until analysis

Melnikov [20]

Plasma

0.2

Retrospective

N/A

N/A

DNAzol reagent (Molecular

Research Center)

Methylation-sensitive

restriction enzyme treatment

BonDurant [21]

Serum

0.5

Retrospective

N/A

N/A

Proteinase K/SDS at 50 °C for 48 h

Phenol/chloroform (twice)

Conventional sodium bisulfite method

Modified DNA stored at 4 °C until analysis

Campan [22]

Serum

0.1

N/A

N/A

Coagulated for 1–4 h, RT

200×g, 10 min, RT

Stored at − 80 °C

QIAamp UltraSens Virus Kit (QIAGEN)

EZ DNA Methylation Kit (Zymo Research)

Häfner [23]

Serum

0.7

N/A

N/A

N/A

NucleoSpin Plasma XS Kit (Macherey–Nagel)

MethylAmp DNA Modification Kit (Epigentek)

Modified DNA stored at − 20 °C until analysis

Liggett [24]

Plasma

0.2

N/A

EDTA

Processed within 2 h after blood draw

1. 2600×g, 10 min, 4 °C

2. 2600×g, 10 min, 4 °C

Stored at − 80 °C

DNAzol BD Reagent (ThermoFisher Scientific)

Methylation-sensitive

restriction enzyme treatment

Dong [25]

Serum

0.5

Prospective

N/A

Coagulated for 2 h at RT

3000×g, 10 min

Stored at − 80 °C

Phenol/chloroform

CpGenome DNA Modification Kit (Chemicon)

Wang [26]

Serum

N/A

Retrospective

N/A

N/A

QIAamp DNA Blood Mini Kit (Qiagen)

EpiTect Bisulfite Kit (QIAGEN)

Zhang [27]

Serum

0.2

Retrospective/prospective

Vacutainer separating gel procoagulant tubes

3500×g , 5 min, 4 °C

Stored at − 80 °C

QIAamp MinElute Virus Spin Kit (QIAGEN)

EpiTect Bisulfite Kit (QIAGEN)

Wu [28]

Plasma

N/A

N/A

N/A

N/A

Proteinase K

Phenol/chloroform

Conventional sodium bisulfite method

Modified DNA stored at − 80 °C until analysis

Zhou [29]

Serum

0.8

N/A

N/A

N/A

Stored at − 70 °C

QIAamp DNA Mini Kit (QIAGEN)

Methylation-sensitive

restriction enzyme treatment

Wang [30]

Serum

N/A

N/A

N/A

3500 rpm/min, 5 min, 4 °C

Stored at − 80 °C

QIAamp DNA Mini Kit (QIAGEN)

N/A

Giannopoulou [31]

Plasma

2

N/A

EDTA

1500×g , 10 min

Stored at − 80 °C

QIAamp Circulating Nucleic Acid Kit (QIAGEN)

EZ DNA Methylation-Gold Kit (Zymo Research)

Modified DNA stored at − 70 °C until analysis

Swellam [32]

Serum

3

N/A

N/A

4000×g , 10 min

Stored at − 80 °C

QIAamp DNA Blood Mini Kit (QIAGEN)

DNA stored at − 80 °C until bisulfite conversion

EpiTect Fast Bisulfite Kit (QIAGEN)

Wang [33]

Serum

0.2

N/A

N/A

3500×g , 5 min, 4 °C

Stored at − 80 °C

QIAamp DNA Blood Mini Kit (QIAGEN)

EpiTect Bisulfite Kit (QIAGEN)

Widschwendter [34]

Serum

1

Prospective

VACUETTE® Z Serum Sep Clot Activator tubes

Processed up to 12–28 h after blood draw

3000 rpm, 10 min

Stored at − 80 °C

Extracted at GATC Biotech (Konstanz, Germany)

Sodium bisulfite conversion at GATC Biotech (Konstanz, Germany)

Giannopoulou [35]

Plasma

2

N/A

EDTA

1500×g, 10 min

Stored at − 80 °C

QIAamp Circulating Nucleic Acid Kit (QIAGEN)

EZ DNA Methylation-Gold Kit (Zymo Research)

Modified DNA stored at − 70 °C until analysis

Dvorská [36]

Plasma

N/A

N/A

N/A

N/A

QIAamp DSP Virus Kit (QIAGEN)

Epitect Bisulfite Kit (QIAGEN)

Modified DNA stored at − 20 °C until analysis

Kumar [37]

Plasma

N/A

N/A

EDTA

3000 rpm, 10 min

Stored at − 80 °C

Proteinase K/SDS at 37 °C overnight

Phenol/chloroform

DNA stored at − 20 °C until bisulfite conversion

EZ DNA Methylation Lightening Kit (Zymo Research)

Liu [11]

Plasma

10

Prospective

Cell-free DNA BCT (Streck)

Processed within 1–5 days after blood draw

QIAamp Circulating Nucleic Acid Kit (QIAGEN)

Automated MagMax Kit (ThermoFisher Scientific)

EZ-96 DNA Methylation Kit (Zymo Research)

Miller [38]

Plasma

1–3.5

N/A

EDTA

N/A

Stored at − 80 °C

NeoGeneStar Cell Free DNA Purification Kit (NeoGeneStar)

EZ DNA Methylation Lightening Kit (Zymo Research)

Singh [39]

Serum

1

N/A

BD Vacutainer® separating gel procoagulation tubes

Processed immediately after blood draw

3500×g, 10 min, 4 °C

Stored at − 80 °C

Proteinase K/SDS

MagMax Cell-Free DNA Magnetic beads extraction (ThermoFisher Scientific)

DNA stored at − 20 °C until bisulfite conversion

Premium Bisulfite Kit (Diagenode)

Modified DNA stored at − 20 °C until analysis

Faaborg [40]

Plasma

1.6–4.1

Retrospective/

prospective

EDTA

Processed within 4 h after blood draw

1. 2000×g, 10 min

2. 10,000×g, 10 min (after thawing of plasma)

Stored at − 80 °C

QIASymphony DSP Circulating DNA (QIAGEN)

EZ DNA Methylation Lightning Kit (Zymo Research)

Miller [41]

Plasma

1.5–4

Retrospective

N/A

N/A

NeoGeneStar Cell Free DNA Purification Kit (NeoGeneStar)

EZ DNA Methylation Lightning Kit (Zymo Research)

Modified DNA stored at − 20 °C until analysis

Singh [42]

Serum

1

N/A

BD Vacutainer separating gel procoagulation tubes

1. 3500×g, 1 min, 4 °C

2. 3500×g, 1 min, 4 °C

Stored at − 80 °C

MagMax Cell-Free DNA Isolation Kit (ThermoFisher Scientific)

Premium Bisulfite Kit (Diagenode)

Modified DNA stored at − 20 °C until analysis

Tserpeli [43]

Plasma

2

N/A

EDTA

1500×g, 10 min

Stored at − 80 °C

QIAamp Circulating Nucleic Acid Kit (QIAGEN)

EZ DNA Methylation Gold Kit (Zymo Research)

Modified DNA stored at − 70 °C until analysis

Marinelli [44]

Plasma

3–6

Retrospective

EDTA

LBgard® Blood Tubes (Biomatrica)

N/A

QIAamp DNA Blood Kit (QIAGEN)

EZ-96 DNA Methylation Kit (Zymo Research)

Tomeva [45]

Plasma

4

Prospective

EDTA

Processed within 1 h after blood draw

1. 2000×g, 10 min, 4–8 °C

2. 16,000 × g, 10 min, 4–8 °C

Stored at − 80 °C until shipment, − 20 °C after arrival

MagMAX Cell-Free DNA Isolation Kit (ThermoFisher Scientific)

KingFisher Duo Prime Magnetic Particle Processor (ThermoFisher Scientific)

DNA stored at − 20 °C until further treatmet

Separation of methylated/unmethylated using MethylMiner™ Methylated DNA Enrichment Kit (Invitrogen, ThermoFisher)