Establishment of a cohort for deep phenotyping of the immune response to influenza vaccination among elderly individuals recruited from the general population

Figures & data

Table 5. Biomaterials collected during the pilot and main studies.

Tube	Amount	Material	Analysis	Pilot	Main
Lithium heparin	3×9 ml	Buffy coat	Immunomonitoring (fresh and cryopreserved cells)	x	—
Serum	7.5 ml	Serum	Serology	x	x
EDTA	1.2 ml	Whole blood	Complete blood count	x	x^*
EDTA	5 ml/7.5 ml	Plasma	Cytokine/chemokine profiling, proteomics, metabolomics, lipidomics	x	x
		Blood without plasma/ Buffy coat	Genomics, transcriptomics
EDTA	5 ml	Whole blood	TCR/BCR profiling	x	—
PAXgene®	2.5 ml	RNA	Transcriptomics, TCR/BCR profiling	x	x
CPT Na-Heparin	2×8 ml	PBMCs	Immunomonitoring (cryopreserved cells)	—	x
TruCulture® (without stimulant)	1 ml	Whole blood	Cytokine/chemokine measurements after 48 h	—	x^**
TruCulture® (with stimulant LPS-SEB)	1 ml	Whole blood	Cytokine/chemokine measurements after 48 h	—	x^**
Stool collection kit (Süsse Stuhlfänger™)	≈2 gr.	Stool	Stool microbiome	—	x^**

* day 0 and 70 only

** day 70 only

Table 4. Duration until sample processing, freezing and processing strategy in the main study.

Sample	Centrifugation	Aliquotation	Incubation	Freezing/storage	Median (mean) time until arrival in biobank (hours)	Median (mean) time until processing (hours)	Median (mean) time until freezing (hours)	Notes
Serum	2000 g, 10 min., 20°C, with brake	≤ 5×500 µl serum	—	Gas phase nitrogen	0:13 (0:17)	0:31 (0:35)	0:46 (0:51)	30–60 min. at room temperature before processing
EDTA	2000 g, 10 min., 20°C, with brake	≤ 7×500 µl plasma1×1000 µl buffy coat	—	Gas phase nitrogen	0:13 (0:17)	0:31 (0:51)	—	—
PAXgene®	—	—	—	After 2 h of incubation at room temperature,−80°C	0:13 (0:17)	No processing	2:00 (2:40)	Freezing after 2 h
CPT	1800 g, 20 min., room temperature	≤ 3×1000 µl PBMCs	—	Controlled-rate −1°C/min cell freezing at −80°C, gas phase nitrogen	0:13 (0:17)	0:41 (0:50)	2:40 (3:26)	—
TruCulture®	—	—	48 h at 37°C	−20°C	0:08 (0:14)	0:10 (0:14)	2 (2) days	Freezing after 48 h of incubation
Stool	—	—	—	−20°C	1.8 (2.1) days	No processing	Freezing directly after receiving	Sample collection at home

Table 1. Descriptive characteristics of the study participants of the pilot and main studies and nonparticipants of the main study, %.

	Participants of the pilot study n = 34	Participants of the main study n = 200	Non-participants of the main study n = 531	p value^**
Sex				0.08
Female	50	43	50
Male	50	57	50
Median age in years (interquartile range)	70 (67–72)	72 (68–76)	73 (69–76)	0.11^***
Body Mass Index^*				0.37
Underweight (≤ 18.49 kg/m^2)	0	1.0	1.6
Normal weight (18.50–24.99 kg/m^2)	35	36	42
Overweight (25.00–29.99 kg/m^2)	44	45	38
Obesity (≥ 30.00 kg/m^2)	21	18	18
Myocardial infarction				0.68
Yes	0	6.0	6.8
No	100	93	91
Don't know	0	0.50	0.38
Missing values	0	0.50	1.7
Cancer				0.54
Yes	21	20	22
No	79	80	76
Don't know	0	0	1.1
Missing values	0	0	1.7
Diabetes mellitus				0.008
Yes	5.9	7.5	15
No	94	92	83
Don't know	0	0.50	0.57
Missing values	0	0	1.7
Self-perceived health status^§				<0.0001
Poor	0	0	0.19
Fair	0	9.0	15
Good	88	64	62
Very good	12	21	14
Excellent	0	4.0	0.19
Missing values	0	2.0	8.0
Ever vaccinated against influenza				0.93
Yes	74	78	78
No	23	21	21
Don't know	2.9	1.0	0.19
Missing values	0	1.5	1.1
Ever vaccinated against pneumococcal infection				NA
Yes	15	24	NA
No	79	68	NA
Don't know	6.1	4.0	NA
Missing values	0	4.0	NA

* Body Mass Index (BMI) was calculated using the formula “weight/height²” (kg/m²). Weight and height were measured at the study center in the pilot and main studies and self-reported by the nonparticipants.

** Chi-square test for comparison between participants and nonparticipants of the main study. The category “Don't know” was not included in the test.

*** Mann-Whitney-U test

§ Assessed with the first question of the SF-36 Health Survey questionnaire.³⁰

NA, not assessed

Table 2. Acceptability of selected study aspects in the pilot and main studies, %.

Study	Items	Strongly agree	Agree	Uncertain	Disagree	Strongly disagree
Pilot study (n = 34)	Time spent for the study was acceptable to me.	100	0	0	0	0
Main study (n = 200)	Time spent for the study was acceptable to me.	83	15	2.0	0	0
Pilot study (n = 34)	The number of blood draws was acceptable to me.	91	5.9	0	2.9	0
Main study (n = 200)	The number of blood draws was acceptable to me.	80	18	1.5	1.0	0
Pilot study (n = 34)	The amount of blood collected was acceptable to me.	94	6.1	0	0	0
Main study (n = 200)	The amount of blood collected was acceptable to me.	80	16	3.0	0.51	0

Table 3. Completeness of biosamples in the pilot and main studies, %.

	Pilot study					Main study
	Proportion of collected samples, %					Proportion of collected samples, %
Biosample types	Day 0n = 34	Day 3^*n = 32	Day 7^*n = 32	Day 21^*n = 32	Day 70n = 34	Day 0n = 200	Day 1/Day 3^**n = 200	Day 7 n = 200	Day 21 n = 200	Day 70^*** n = 139
Serum	100	100	100	96.9	100	100	99.5	100	100	100
Plasma	100	100	96.9	96.9	100	100	99.5	100	100	100
PAXgene®	100	100	100	96.9	100	100	99.5	100	100	100

* Two participants were in Group A with 2 study visits (on day 0 and day 70).

** Half of the participants were invited for day 1 and half for day 3.

*** A subsample of 139 individuals were invited for day 70 (see Main study).

Figure 1. Design of the pilot study (A) and main study (B) – 2 prospective, population-based studies from December 2014 to May 2015 (pilot) and September 2015 to May 2016 (main). *Half of the study participants were invited for day 1 and half for day 3.