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
0 1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899
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?