mrepo:topics:biologicalsamples [MRC Lifelong Health & Ageing Unit at UCL]

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Biological Samples

Age 53

Biological samples were collected for the first time in 1999; non-fasting blood samples were used to measure indicators of risk factors for cardiovascular disease (cholesterol, HDL, LDL, triglyceride and glycoslated haemoglobin) and nutritional markers (red cell folate, plasma folate, plasma vitamin B12 and plasma ferritin).

The samples also include a blood sample and a cheek swab from which DNA was derived. A further sample was taken and stored for future cell line cultures. Blood was taken using standard phlebotomy techniques using the BD vacutainer system.

Stored biological samples available from 1999 comprise of EDTA plasma, lithium heparin plasma, cell lines, and DNA. The following tables list the current data available from these samples.

Order of Collection	Blood Tube	Analytes (destination)	Repository aliquots	Comment
1	Buccal swabs	DNA (MRC-HBGU)	DNA	DNA quantified by Genprobe
2	1 x 3ml EDTA tube	DNA (MRC-HBGU)	DNA	Limited availability
3	1 x 2ml EDTA tube	DNA (MRC-HBGU)	DNA	Limited availability
4	1 x 6ml ACD tube	ECACC	ECACC	EBV transformed lymphoid cell lines created
5	1 x 4ml Lithium heparin tube	Cholesterol, HDL cholesterol, triglyceride (RVI)	Lithium heparin	Limited availability
6	1 x 3ml EDTA tube	Red cell folate, plasma folate, plasma B12, plasma ferritin (RVI)	EDTA plasma	Limited availability

Age 60-64

Blood

At 60-64 years, fasting blood, saliva and overnight urine samples were collected, either in Clinical Research Facilities or at home. The volume of blood and processing protocol was designed to provide aliquots suitable for the analysis of biomarkers identified by LHA and its collaborators to be relevant to ageing and health. The collection and storage of the biological samples was organised by MRC Human Nutrition Research (HNR) in Cambridge.

Up to 55ml of fasting blood was obtained from the study members, with a smaller volume being collected at remote home visits because the time to processing was too long to maintain the integrity of some analytes. For example, potassium, creatinine and phosphate results were excluded from the reports of samples collected during remote home visits as the overnight delay before separation meant the result would not reflect the in vivo concentrations of these analytes.

DNA extractions were conducted on the EDTA tube used in the first round blood haematology analysis. Samples for lymphoid cell line transformations were originally collected in 1999; for those that did not previously provide a sample, one was collected in 2006-10.

Order of collection priority	Blood Tube	Aliquots for first round blood analysis	Repository aliquots
1	1 x 2.7ml EDTA tube (EN1)	None as sample was sent for haematology analysis	EN1 generated for DNA extraction
2	1 x 2.7ml EDTA tube (EN2)	None as sample was sent for HbA1c and red cell folate analysis	None
3	1 x 2.7 Fluoride tube (FL N1)	FLA1 for glucose analysis	None
4*	1 x 7.5ml Lithium heparin tube (trace metal) (LH N1)	LH INS for insulin; LH VITC for vitamin C analysis	LH B1, LH C1, LH C2, LH C3
5	1 x 85ml CPDA (for cell lines if required) (CP N1)	None as sent to ECACC for cell lines	None
6	1 x 7.5ml Serum tube (SE N1)	Samples from the two serum tubes were used to produce priority aliquots SE A1 for TFTs and SE A2 for analyses at NHR	SE B1, SE C1, SE C2
7*	1 x 7.5ml Serum tube (SE N2)	Samples from the two serum tubes were used to produce priority aliquots SE A1 for TFTs and SE A2 for analyses at NHR	SE B1, SE C1, SE C2
8*	1 x 4.9ml EDTA tube (E N3)	None	E B1, E C1
8*	1 x 10ml Citrate tube (CI N1)	None	CI B1, CI C1, CI C2

*Blood tubes/aliquots not collected at remote home visits

Urine

Overnight urine was collected, however the protocol for processing and storing the samples was changed during the data collection, resulting in a different number of aliquots being collected compared to the later period. The original protocol was inappropriate for albumin measurement as they should have been centrifuged; and they were stored at -20ºC instead of -80ºC. The change in protocol resulted in an additional aliquot of urine being centrifuged and stored at -80ºC. The date of collection indicated whether the revised protocol was used or not.

Saliva

The collection of saliva samples only began after a pilot study, and the protocol consisted of one sample collected on the day of the clinical assessment and a further three at home. HNR received all of these first samples, but for about 68 people (3%) no further samples were received. The collected saliva samples were sent to Germany for cortisol analysis before being returned to HNR.

Age 68-70

At 68-70 years non-fasting blood samples were collected at a home visit by research nurses. Prior to posting off for analysis, serum samples were allowed to clot for 15 minutes before being spun. A number of biomarkers were measured: haemoglobin, HbA1C, total-, LDL-, HDL- cholesterol, triglycerides.

A number of assays have been undertaken and the remaining aliquots of EDTA, citrate plasma and serum are stored at the Bristol Bioresource Laboratories. DNA extractions have been conducted at MRC Epidemiology, Cambridge, as per the 2006-10 samples.

Biomarkers available since age 53. Biomarkers highlighted in bold are not present in other UK birth cohorts (as far as is known)

System	Biomarkers at 53 years	Biomarkers at 60-64 years	Biomarkers at 68-70 years
Metabolic		Adiponectin (μg/mL)
	Cholesterol, Total (mmol/L)	Cholesterol, Total (mmol/L)	Cholesterol, Total (mmol/L)
	Cholesterol, High density Lipoprotein, LDL (mmol/L)	Cholesterol, High density Lipoprotein, HDL (mmol/L)	Cholesterol, High density Lipoprotein, HDL (mmol/L)
	Cholesterol, Low density Lipoprotein, LDL (mmol/L)	Cholesterol, Low density Lipoprotein, LDL (mmol/L)	Cholesterol, Low density Lipoprotein, LDL (mmol/L)
	Triglycerides (mmol/L)	Triglycerides (mmol/L)	Triglycerides (mmol/L)
		Glucose (nmol/L)
		Insulin (pmol/L)
		Leptin (ng/mL)
		Lipoprotein[a] (mg/dL)
		Proinsulin (pmol/L)
	Glycated Haemoglobin, HbA1c (mmol/mol)	Glycated Haemoglobin, HbA1c (mmol/mol)	Glycated Haemoglobin, HbA1c (mmol/mol)
Diet & Iron Markers		Ferritin (ng/mL)
		Haemoglobin, Hb (g/L)	Haemoglobin, Hb (g/L)
		Iron (μg/dL)
		Vitamin C (μmol/L)
		Vitamin D3 (250HD3) (ng/mL)†
Inflammatory		Anti-Thyroperoxidase, TPO (IU/mL)
		Basophils (x10⁹/L)	Basophils (x10⁹/L)
		C-Reactive Protein, CRP (mg/L)
		Eosinophils (x10⁹/L)	Eosinophils (x10⁹/L)
		Fibrinogen (g/L)
		Homocysteine concentration (μmol/L)
		Interleukin-6, IL-6 (pg/mL)
		Tissue Plasminogen Activator, TPA (ng/mL)
		Von Willebrand Factor, VWF (IU/dL)
		Red Cell Folate, RCF (nmol/L)
		Lymphocytes (x10⁹/L)	Lymphocytes (x10⁹/L)
		Mean Corpuscular Volume, MCV
		Mean Corpuscular Haemoglobin, MCH
		Mean Platelet Volume, MPV
		Monocytes (x10⁹/L)	Monocytes (x10⁹/L)
		Neutrophils (x10⁹/L)	Neutrophils (x10⁹/L)
		Platelet count (x10⁹/L)	Platelet count (x10⁹/L)
		Platelet Distribution Width, PDW (%)
		Potassium (mmol/L)
		Protein (blood), Total (g/L)
		Red Blood Cell, RBC, count (x10¹²/L)	Red Blood Cell, RBC, count (x10¹²/L)
		White Blood Cell, WBC, count (x10⁹/L)	White Blood Cell, WBC, count (x10⁹/L)
Neuro-Endocrine		Cortisol (saliva) (nmol/L)
	DHEAS (umol/L)	DHEAS (umol/L)
	Insulin Growth Factor 1, IGF-1 (nmol/L)	Insulin Growth Factor 1, IGF-1 (nmol/L)
		Insulin Growth Factor 2, IGF-2 (nmol/L)
		Insulin Growth Factor IGF-BP3 (ng/mL)
	Sex hormone binding globulin, SHBG (nmol/L)	Sex hormone binding globulin, SHBG (nmol/L)
	Testosterone (nmol/L)	Testosterone (nmol/L)
		Thyroid tests (T3, T4) (pmol/L)
		Thyroid Stimulating Hormone, TSH (mU/L)
Kidney & Liver		Calcium (mmol/L)
		Creatinine (blood, urine) (mmol/L)
		Phosphate (mmol/L)
		Urea (nmol/L)
		Alanine Transaminase , ALT (IU/L)
		Alkaline Phosphatase, ALP (IU/L)
		Aspartate Transaminase, AST (IU/L)
		Albumin (blood, urine) (mg/L)
		Bilirubin (mg/dL)
		Cystatin C (mg/L)
		Gamma Glutamyl Transferase, GGT (IU/L)
		Glucose (urine) (dipstick) (mmol/L)
		Globulin (mg/dL)
		Ketones (urine) (dipstick) (positive/negative)
		Liver Iron Concentration, LIC (%)
		Potassium (serum) (mmol/L)
		Potassium (urine) (mmol/L)
		Protein (urine) (positive/negative)
		Sodium (urine) (mmol/L)
		Sodium (blood) (mmol/L)
		Urate (g/L)