Table 1.

Protocol variability in breast cancer-associated biomarker discovery workflows.

Aim	Pre-Analytical Phase						Analytical Phase		Post-Analytical Phase	Ref
	BioSource	Collection Tube	Time to Sample Processing	Centrifugation	Storage	Tumour Grade	Technique	Validation Method	Hypothesis Test Performed
Proteomic	Serum	NA	4 °C for 1–2 h	3000 rpm for 5 min + 12,000 rpm for 5 min	−80 °C	NA	SELDI-TOF-MS	SDS-PAGE MALDI-TOF/TOF	t-test ANOVA	[26]
	Serum	Plastic tube with clot activator	15 min	3280× g for 5 min, 4 °C	−80 °C	NA	SELDI-TOF MALDI-TOF-TOF	NA	t-tests Multivariate discrimination analysis ANOVA ANN ROC	[27]
	Plasma	K2EDTA tube	2 h	1300× g for 10 min	−80 °C	NA	1D gel electrophoresis 2D gel electrophoresis LC-MS/MS	WB	Unpaired t-test	[28]
	Plasma	EDTA tube	30 min	4000× g for 30 min	−80 °C	NA	LC-MS/MS	WB	t-test	[29]
	Plasma	Sodium EDTA tube	NA	1400× g for 5 min, 4 °C	ND	Low and high grade	Label-free nano-LC/MSMS	WB	Mann–Whitney	[30]
	NAF	Graduated micropipette	Immediately	1500 rpm for 10 min	−80 °C	I/II	SELDI-TOF-MS	ELISA	Supervised and unsupervised cluster analysis	[14]
	NAF	Tube pre-treated with cocktail mixture of protease inhibitor	<30 min	NA	ST: −20 °C LT: −80 °C	I–III	1D LC-MS/MS	NA	Pearson’s correlation coefficients Paired Student t-test	[31]
	Urine	Sterile tube	Immediately	2000× g for 10 min, 4 °C	ST: −20 °C LT: −80 °C	II–III	Label-free LC-MS/MS	WB	ANOVA	[9]
	First Morning Urine	Tube containing 0.02% w/v Sodium Azide)	NA	NA	ND	I/II	Standardisation phase: 2D gel electrophoresis Discovery phase: 2D-DIGE, MALDI-TOF-TOF, SWATH-MS, iTRAQ, LC-QTOF	WB MRM	Supervised and unsupervised cluster analysis Multivariate analysis Chi-square	[10]
Metabolomic	Plasma	EDTA tube	<2 h	3000× g for 10 min, 4 °C	−80 °C	I–III	LC-MS	NA	Kruskal–Wallis Mann–Whitney U test ROC	[32]
	Plasma	K2EDTA tube	Immediately	1500× g for 10 min, RT	−80 °C	I–III	LC-QTOF-MS LC-QQQ-MS	NA	Student’s t-test PLS-DA OPLS-DA	[33]
	Serum	Vacutainer tube	30 min	3000 rpm for 10 min, 4 °C	−80 °C	I–III	UHPLC-QTOF-(ESIþ)-MS	NA	Pearson ROC PCA PLS-DA t-test	[34]
	First Morning Urine	NA	NA	3000× g for 10 min, RT	−80 °C	I/III	GC–MS LC-QTOF/MS	NA	PCA OPLS-DA Univariate analysis Unpaired t-test Mann–Whitney U test	[35]
	Saliva	Polypropylene tube	NA	NA	−80 °C	0–IV	CE-TOF-MS	LC-QQQ-MS	Mann–Whitney U test Kruskal–Wallis Multiple logistic regression Multiple AD tree models	[36]
	Saliva	NA	10 min	13,500 rpm for 20 min, 4 °C	−40 °C	I–IV	HILIC-ESI-MS RPLC-ESI-MS	NA	Mann–Whitney U test PLS-DA PCA	[37]
Lipidomic	Plasma	Heparin tube	NA	1500× g for 15 min	−80 °C	I/II	UPLC-QTOF/MS	NA	t-test One-way ANOVA OPLS-D	[38]
	Plasma	EDTA tube	<2 h	2600× g for 10 min, 4 °C	−80 °C	0- II	LC-ESI-MS/MS	NA	t-test Binary logical regression ROC	[39]
	Serum	NA	NA	NA	−80 °C	NA	NMR spectroscopy	NA	t-test Mann–Whitney U test Chi-square Binary logistic regression	[40]
	First Morning Urine	NA	NA	3000× g for 10 min, RT	−80 °C	I/III	LC–MS	NA	OPLS-DA Univariate analysis Unpaired t-test Mann–Whitney U test ROC	[35]
	Saliva	Polypropylene tube	NA	10,000× g for 10 min	Without freezing and storage	I–III	IR spectroscopy	NA	Mann–Whitney U-test Kruskal–Wallis Multivariate comparison	[41]

NA: Not Available, EDTA: Ethylenediaminetetraacetic Acid, K2 EDTA: Dipotassium Ethylenediaminetetraacetic Acid, h: Hour, min: Minute, RT: Room Temperature, ST: Short-Term, LT: Long-Term, WB: Western Blotting, ELISA: Enzyme-linked Immunosorbent Assay, ANN: Artificial Neural Network, ROC: Receiver Operating Characteristic.