Blood samples for diabetes research- Anything to consider before starting -OMICS

investigations?

Rainer LehmannInstitute for Clinical Chemistry and Pathobiochemistry, University Hospital Tuebingen

andDept. of Molecular Diabetology and Core Facility DZD Clinical Chemistry Laboratory

Inst. for Diabetes Research and Metabolic Diseases (HMGU Tuebingen/Munich)

German Center for Diabetes Research (DZD)Germany

- My daily routine –The Clinical Chemistry Central Laboratory

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Probeneingang im Zentrallabor

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Probeneingang im ZentrallaborAnalysis per day (24 h)5000

4000

3000

2000

1000

h

n=

● ca. 6.5 mio. tests/year

● ca. 4300 samples daily

● 24/7 laboratory

● 42 staff members (incl. research)(18 MTAs on shift duty)

● accredited (DIN EN ISO 15189)

Are highly accurate measurements the most significant steps in –omics-driven

diabetes research?

YES and NO

- The samples in diabetes research studies -

?

in diabetes research

projects

blood samples end up in

uniform serum and plasma

aliquots

- The samples in diabetes research studies -

… and are subsequentlystored for months or yearsat high costs in lab freezersor biobanks

-omics analysesand other

investigations

- The samples in biomedical research studies -

Biobank Bern

poorsample quality

metabolomicsanalysis bioinformatics

poor / misleading

results

garbage in, garbage out research

Possible Consequences for –omics investigations

The sample quality is also very important!

Are highly accurate measurements the most significant steps in –omics-driven

diabetes research?

Considerations related to sample quality and study outcome in diabetes research

#1 Before sample collection

#2 Crucial aspects of the sample collection process

#3 Control of sample quality

#4 Sample storage / biobanking

Outline

Consideration #1

Before Sample Collection

Plasma or Serum,

which material is preferable?

PlasmaSerum

Plasma / Serum

Anti-coagulants

can beimmediatlycentrifugedor cooled

Plasma

Blood

clottedin an uprightpostion

clottedwhile lyingon a desk

Serum

keep for30 – 60 min

undisturbed at room

temperature

centrifugeor cool

What are the consequences for –omics analysis caused bythe

a) clotting process (incl. activation of thrombocytes)b) required exposure to room temperature

???

Serum

Special characteristics of SERUM samples

Liu X., et al. Anal. Chim. Acta, 1037: 293, 2018

serum : 60 min clotting w/o coag. enhancers

Plasma Serum

99 out of 216 lipids /metabolites were sign. different

Plasma vs serumlipidome/metabolome

During blood coagulation platelets, leukocytes and erythrocytes release

various compounds into serum, including enzymes and other proteins,

metabolites, lipids, …

the coagulation process leads ex vivo to the degradation of proteins,

as well as to the generation or modification of lipid species and other

metabolites

Special characteristics of SERUM samples

For several reasons we and many others do not recommend serum for –omics analysis!

We prefer and recommend plasma, but…..

plasma serum

99 of 216 metabolites were sign. different

(diff. > 10%)

standard plasma is not cell free!( 1ml = ca. 27 000 000 platelets)

standard plasma

platelet-free plasma

centrif: 4000 g / 10´/ 15°C

Plasma vs Serum

A = standard plasma

B – D = platelet-free plasma

Standard Plasma vs Platelet-free plasma

n = 10

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

Num

ber o

f pla

tele

ts p

er μL

A B C D

Detected mean platelet count in common and platlet-free plasma

4

3 2

18

107

695

condition: A ○ B □ C ◆ D ▼

PCA scores plot of standard vs platelet-free plasma

Liu X., et al. Anal. Chim. Acta, 1037: 293-300, 2018

Plasma:Centrifugation force is crucial

1500 × g 10 min vs. 3000 × g 5 min

137,500,000 plt/ml plasma

59,000,000 plt/ml plasma

differences in the plasma metabolome

Comparison of two routine centrifugation

forces

4000 × g 10 min

27,000,000 plt/ml plasma

Liu X., et al. 2018

Plasma is recommended as sample material of choice for high-resolution -omics investigations

for lipidomics/metabolomics: spray-coated EDTA plasma

Take home message

Centrifugation forces for whole blood are critical:

low centrifugation forces or short centrifugation times may lead to residual

platelets in the plasma, which may affect results of –omics studies

e.g. the plasma levels of proteins or lipids abundant in platelets

too high centrifugation forces (> 4500g) may cause hemolytic plasma or serum

preparations and alter the metabolite (incl. lipids) and protein levels

Consideration #2Crucial Aspects of the Sample Collection

Process

The pre-analytical phase

pre-analytical process

biobanktransport analysiscentrifugation plasma /serum

samplepreparation

Analytical specialists (wet lab)

data anaylsis / bioinformatics

validresults

clinicalstudy

bloodcollection

hospital research laboratory

In the clinic the interest in the pre-analytical phase in general is minor, because patient care is more important

In complex diabetes studies blood collection is often entitled as the easy part

The pre-analytical phase

pre-analytical process

biobanktransport analysiscentrifugation plasma /serum

samplepreparation

Analytical specialists (wet lab)

data anaylsis / bioinformatics

validresults

clinicalstudy

bloodcollection

hospital research laboratory

The centrifuge was in another building. We could not process the blood samples before 2 h, is this a problem?

Typical questions of investigators (frequently asked after the end of their studies) are:

Several biobank samples of my study have been thawed one or two times. Are they still suitable e.g. for lipidomics/metabolomics investigations?

One third of the blood of our study was drawn by inexperienced medical students. Unfortunately this resulted in several more or less hemolytic samples. Can we still use the sample set for –omics analysis?

....

The centrifuge of our ward was highly frequented, consequently the processing of some blood samples was delayed. Is this a problem for –omics analysis?

biobanktransport centrifugation plasma /serum

clinicalstudy

bloodcollection

The pre-analytical phase

the period from blood collection until separation of blood cells from

plasma/serum = “time-to-processing”

exposure of whole blood to room temperature

The most critical step in the processing of blood samples

pre-analytical process

biobanktransport analysiscentrifugation plasma /serum

samplepreparation

Analytical specialists (wet lab)

data anaylsis / bioinformatics

validresults

clinicalstudy

bloodcollection

The pre-analytical phase

No problem! …we apply a sophisticated SOP!

…prolonged exposure of blood to room temperature is excluded!

The pre-analytical phase

non-targeted metabolomics

2h, 4h, 8h and 24h

Experimental design

Simulation of clinic internal handling / transportation 2h, 4h, 8h and 24h exposure of blood to room temperature or iced water

Processing of whole blood

0h= processed

at once

Exposure of EDTA blood samples to room temperature

Transportation / processing of bloodfo

ld c

hang

e of

pea

k ar

ea

0h

2h

4h

8h

24h

0.1

1.0

10(m

/z)

external transports

time needed for clinical internal handling / transport

P. YinA. Peter

Yin P, Peter A., et al. Clin. Chem. 2013

n = 10

exposure of whole blood samples to room temperature lead to pronounced changes of metabolite levels, even after 2h

Consequence:Drawing, handling and further processing of blood must follow a

sophisticated, meaningful, but feasible SOP

a too strict SOP is highly error prone in a clinical setting

(e.g. to stipulate centrifugation of blood within 15 min)

Hemolysis

The pre-analytical phase

pre-analytical process

biobanktransport analysiscentrifugation plasma /serum

samplepreparation

Analytical specialists (wet lab)

data anaylsis / bioinformatics

validresults

clinicalstudy

bloodcollection

no moderate severe

Hemolysis

Hemolytic specimens occur frequently in clinical laboratories as high as 3.3% of all of daily routine samples are more or less hemolytic

G. Lippi et al. CCLM (2008)

this means e.g. around 100 – 130 samples / day in our lab

hemolytic samples may be collected and also be included samples sets stored in freezers/biobanks for biomarker studies

Data from a non-targeted metabolomics approach

18% of all metabolite ion masseswere significantly altered (p<0.05, FDR<0.1)

moderate hemolysis

severe hemolysis

no hemolysis

Intensity

High

Yin P, Peter A, et al. Clin. Chem. 2013

P. YinA. Peter

Hemolysis

Take home message

- hemolytic samples should be excluded, at least from high resolution -omicsresearch projects

- lipids/metabolites affected by hemolysis (Trp, LPC C16:0,….) must be thoroughly validated for robustness before nominated as biomarker candidates in research studies

and

- all plasma/serum samples should be tested for hemolysis before use or storage

free Hb can easily be measured by a two wavelenght methodGolf-SW et al. J Clin Chem Clin Biochem (1985)

Hemolysis

Consideration #3

Sample quality control

What are the currently applied strategies to

control the quality of blood samples???

HIL check

SOPs

protocols of sample handling

…. but no valid evaluation of the real sample quality is applied

We aimed to find a biomarker

detecting the prolonged exposure

of whole blood to room temperature

Study Design

2h 4hexposure to

room temperature

2h 4hin iced water

0h

Biomarker screening in plasma (non-targeted metabolomics)(covering >1800 metabolite ion features)

targeted analysis of the QC-biomarker Robustness tests / Validation in plasma and serum (n > 2000)

whole blood whole blood

Liu X, Hoene M, et al. Clin. Chem. 2018

processed at once

Non-targeted analysis

detected features：1843 (pos.: 926; neg.: 917; using an SCIEX triple TOF-MS)

Significant changed features in comparison to freshly prepared blood (changes in signal intensity > 10% were respected)

2 h 4 hroom temp. 9.2 % 16.1 %iced water 0.5 % 1.8 %

Metabolite stability in whole bloodat room temperature and in iced water (4°C)

Liu X, Hoene M, et al. Clin. Chem. 2018

n= 30 samples / time point

A biomarker reflecting exposure ofwhole blood to room temperature

Liu X, Hoene M, et al. Clin. Chem. 2018

(4E,14Z)-sphingadienine-C18-1-phosphate(S1P d18:2)

(n= 79 samples / time point)

Targeted analysis of the QC biomarker

Biobank samples (n > 1400)- 11 biobanks

- China, France, Germany

Practicability test

Validation of the QC-marker

0

0.0567

0.1134

0.1701

0.2268

0.2835

0.3402

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Study participant number

good sample quality = up to 4h exposure of whole blood in iced water (4°C) before

centrifugation and separation of plasma from blood cells

μg/ml

goodquality

Definition of cutoff

Important note:Plasma or serum samples above the cut-off are still useful for the measurement of robust parameters (like liver enzymes, albumin, cortisol, haptoglobin, etc.) !!!!!!!

Validation of the QC-markers

= cut-off for good serum sample quality

collected by different persons in a clinic setting under daily routine conditions

according toSOPs

S1P d18:2 levels in biobank samples collected according to SOPs

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Biobank 4: Serum samples

Study 1 (n=30) Study 2 (n=30) Study 3 (n=20)

S1P d18:2μg/mL

collected by one specialized team at one site in the same hospital

according to a SOP0.00

0.050.100.150.200.250.300.350.400.450.50 Biobank 4: Serum samples

Study 4 (n=113)

μg/mL

Delayed handling and processing of blood samples before centrifugation

can be assessed without knowledge of the preanalytical process using

S1P d18:2 as QC-marker

Take home message

Consideration #4Sample storage

Example:Repetitive freeze and thaw cycles (Biobanking)

Problem: the number of aliquots is limited leads unavoidable to repetitive freeze and thaw

cycles aiming to save sample material

we investigated EDTA plasma by non-targeted metabolomics:

controls (metabolomics sample pretreatment was performed at once after drawing blood)

1x frozen (= common biobank sample)

2x thawed / frozen

4 x thawed / frozen

Repetitive freeze/thaw cycles

Yin P, Peter A, et al. Clin Chem 2013; 59:833-45

P. YinA. Peter

no significant alterations of metabolite ion masses between fresh controls and 1x frozen plasma samples were detected

unexpectedly, only < 1% of the masses changed significantly after 2x and 4x freeze/thaw cycles!

Repetitive freeze/thaw cycles

we detected individual differences in the sample stability! instabilities of the metabolite pattern were detected in plasma samples of 2 out of 10 individuals, even in 1x frozen aliquots!

Suggestions for handling of stored samples:- non refrozen plasma aliquots are recommended for –omics

analysis

- mix-up of samples exposed to different freeze and thaw cycles should be avoided

Take home message

FinalBrief Summary

Blood collection is the easy part of complex diabetes studies,BUTmay heavily affect the quality of samples and consequently the outcome and success of –omics results but possibly also beyond

Sample collection

Poor sample quality, caused by delayed handling and processing of blood

samples before centrifugation, can be assessed using S1P d18:2

SOPsare valuable tools to regulate pre-analytical processes

BUT

the recommendations may vary from SOP to SOP (multi-centerstudies!), e.g. based on logistical limitations and subsequent compromises in the SOP

SOPs need to be meaningful, but also feasible!

a too strict SOP is highly error prone in a clinical setting (e.g. to stipulate centrifugation of blood within 15 min)

Standard Operating Procedure (SOP)

Guowang XuXinjie ZhaoPerry Yin

Chunxiu HuXiaolin Wang

Xiaoli HuJia Li

Xinyu Liu

Hans-Ulrich HäringAndreas Peter

Andreas Fritsche Norbert StefanMichael Haap

Robert WagnerLouise Fritsche

Erwin SchleicherAnn K. Horlacher

Heike Runge Clea Kline

Miriam HoeneSabine Neukamm

Lisa KapplerAndreas NiessCora Weigert

Bente K. Pedersen Peter Plomgaard

Jakob Hansen Xiaohui LinHai Wei

Dalian University of Technology

Thank you for your attention!

Dalian Instituteof Chemical Physics

- CAS -

Points to be considered before sample collection - tubes: test and define tubes for body fluid collection, sample preparation, storage (do the labels resist to storage conditions? Printed/handwritten labels or pre-labelled 2D barcodes?)) important that the same anticoagulant (ideally the same tubes, same brand) are used

throughout the study and among studies that will be compared- tips and cups for sample preparation: are they chemical resistant to the applied procedures

plasticizer!- serum or plasma? …same clotting procedure? …plasma, which additive?- selection and definition of centrifugation forces- planned sample collection: day time, pre- or postprandial state, etc.- tube labeling: harmonize labeling for all participating sites- sample aliquots for long-term storage: define volume and number by the analytical needs- multicenter studies (same SOP?, same collection tubes? etc. test all tubes if different brands are used in the participating hospitals)

- health state of control subjects: perform simple test and record results ( “apparently healthy individuals” … e.g. measure hsCRP, creatinine, etc.)

- data storage: structure (e.g. file and sample name), access regulations, back-up

Keywords to achieve high quality samples in clinical studies

Potential limitations affecting sample quality

points for a pre-analytical check list in the clinic / study ward- frequently changing staff responsible for sample collection? Experienced staff or e.g. students?- distraction during sample collection/handling by clinical routine duties (phone call, patients,…)- room temperature during sample collection/handling (air conditioned, not regulated)?- cooling option for body fluids nearby (ice machine, refrigerator,…?)- distance / time to centrifuge at the site of sample collection- is the centrifuge heavily frequented?- storage option for body fluids nearby (+4°C, -20°C, -80°C, liquid nitrogen)?- transportation of samples (conditions, time, temperature)?- options to check the sample quality?- possibilities to check the health state of study participants by routine parameters?