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Rainer Lehmann, University Hospital Tübingen, Germany
Markers of biobank sample quality
Symposium 4Biospecimen Science
Rainer LehmannDiv. of Clinical Chemistry and Pathobiochemistry
University Hospital Tuebingenand
Institute for Diabetes Research and Metabolic DiseasesGerman Center for Diabetes Research (DZD)
Germany
Rainer Lehmann, University Hospital Tübingen, Germany
- good experimental design
- precise, robust and reproducible analytical techniques (wet lab)
- sophisticated data analysis (dry lab) and interpretation
The general opinion is, that essential pre-requisites for the success of research projects are
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Rainer Lehmann, University Hospital Tübingen, Germany
„….the quality of biospecimen is of paramount importancefor the reliability and validity of research results and all downstream applications.“ Fay Betsou
(ISBER President 2013-2014)
Biobank sample quality
pre-analytical phase!
Rainer Lehmann, University Hospital Tübingen, Germany
Unfortunately, in general the interest of many investigators for the pre-analytical phase is only minor
In complex clinical studies blood collection is often entitled as the easy part
Biobanking / Blood collection
transport
centri-fugation
plasma / serum
clinicalstudy
bloodcollection freezer
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Rainer Lehmann, University Hospital Tübingen, Germany
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 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?
Biobanking / Blood collection
....
The centrifuge of our ward was highly frequented, consequently the processing of some blood samples was delayed. Is this a problem?
transport
centri-fugation
plasma / serum
clinicalstudy
bloodcollection freezer
Rainer Lehmann, University Hospital Tübingen, Germany
The centrifuge was in another building. I could not process the blood samples before 2 h, is this a problem?
Typical questions of investigators (frequently 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 metabolomics investigations?
One third of the blood of the study was drawn by medical students, which unfortunately resulted in several more or less hemolytic samples. Can we use the samples?
Biobanking / Blood collection
....
The centrifuge of our ward was highly frequented, that is way the processing of some samples was delayed.Variable and prolongated exposure time of blood to room temperature before centrifugation in occasional instances
Hemolytic sera/plasma are in the sample set
Repetitive freeze and thaw cycles had been performed with some samples
Systematic prolongation of the exposure time of blood before processing
transport
centri-fugation
plasma / serum
clinicalstudy
bloodcollection freezer
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Rainer Lehmann, University Hospital Tübingen, Germany
Markers of biobank sample quality
Blood collection Transportation Storage
hemolysis delayed processingrepetitive
freeze/thaw cycles
we applied metabolomics to investigated these preanalyticalsteps
Rainer Lehmann, University Hospital Tübingen, Germany
Metabolomics
NON-TARGETED MetabolomicsTARGETED Metabolomics
analysis of selected, well-knownmetabolites
20 - 300 metabolites per sample
automation (commercial kits available)
quantitative
population based studies
> 500 samples / week possible
2 weeks until final result
non-hypothesis driven, comprehensive approach
> 2.000 metabolite-ion masses per sample
time consuming and labor intensive
semi-quantitative
well selected and characterized samples
≥ 4 months until final result
identification of most important metabolites
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Rainer Lehmann, University Hospital Tübingen, Germany
primary note for the coming slides: Scheme of data evalution
Metabolomics data processing
Rainer Lehmann, University Hospital Tübingen, Germany
blood collection / hemolysis
transportation
storageHemolytic specimens occur frequently in clinical laboratories
as high as 3.3% of all of the routine samples,that means e.g. around 100 – 130 samples / day in our lab
they account for up to 40%–70% of all unsuitable specimens identifiedG. Lippi et al. CCLM (2008)
pre-analytical process Analytical specialists (wet lab)
Markers of biobank sample quality
biobank
analysis
samplepreparation
dataevaluation /
bioinformaticsvalid results
transport
centri-fugation
plasma / serum
clinicalstudy
bloodcollection
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Rainer Lehmann, University Hospital Tübingen, Germany
Blood drawing
Hemolysis
n = 10
non-targeted metabolomics
classical parameters
blooddrawing
Rainer Lehmann, University Hospital Tübingen, Germany
Blood drawing
no hemolysis
moderate hemolysis
strong hemolysis
18% of all metabolite ion masses were significantly altered (p<0.05, FDR<0.1)
Intensity
High
HemolysisPCA scores plot
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Rainer Lehmann, University Hospital Tübingen, Germany
nontargeted Metabolomics
Crucial point in non-targeted metabolomics
Bioinformatics canditate list of features / ion masses
Elucidation of the metabolite identity
Data base searches / MSn / confirmation by a standard compound
Rainer Lehmann, University Hospital Tübingen, Germany
Blood drawing
Identified metabolites (sign. changed):Lyso-PC 16:0 Lyso-PC 18:0 Lyso-PC 18:1TryptophanSphingosine 1-phosphate (S-1-P)N-acetylornithineC8-carnitine
Suggestions:- hemolytic samples should be excluded, at least from high resolution -omics
research projects
- S-1-P, Trp, LPC C16:0,…. should only carefully be nominated as biomarker candidates in research studies and must be thoroughly validated for robustness
- All biobank samples should be tested for hemolysis before storagefree Hb can easily be measured by a two wavelenght method
Golf-SW et al. J Clin Chem Clin Biochem (1985)
Hemolysis
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Rainer Lehmann, University Hospital Tübingen, Germany
blood collection
transportation / processing of blood
storage
Markers of biobank sample quality
biobank
analysis
samplepreparation
dataevaluation /
bioinformaticsvalid results
transport
centri-fugation
plasma / serum
clinicalstudy
bloodcollection
Rainer Lehmann, University Hospital Tübingen, Germany
© Universitätsklinikum Ulm
A 9 ml sample of blood from an adult subject contains
5 × 1010 red blood cells
3 × 109 platelets
8 × 107 leukocytes
„liquid tissue“
Markers of biobank sample quality
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Rainer Lehmann, University Hospital Tübingen, Germany
Transportation / processing of blood
n = 10
non-targeted metabolomics by LC/MS
n = 10
2h, 4h, 8h and 24h 2h and 4h
Experimental design- 2h to 4 h exposure of blood to room temperature or ice water
clinic internal handling / transportation
- 8h and 24 h exposure to room temperature external transportation
Rainer Lehmann, University Hospital Tübingen, Germany
1
2
3
45
6
79
10
-70
-60
-50
-40
-30
-20
-10
0
10
20
30
40
50
60
70
80
-100 -80 -60 -40 -20 0 20 40 60 80 100
8
control2 h4 h
ice water
-20
-10
0
10
20
-40 -30 -20 -10 0 10 20 30 40SIMCA-P 11 - 2012/1/10 11:29:47
room temperature
Transport of whole blood
(EDTA-blood)
n = 10n = 10
Yin P, ….Xu G., Lehmann R.: Clin Chem 2013; 59:833-45
Transportation / processing of blood
Peiyuan Yin
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Rainer Lehmann, University Hospital Tübingen, Germany Yin P, ….Xu G., Lehmann R.: Clin Chem 2013; 59:833-45
Exposure of EDTA blood samples to room temperature
fold cha
nge of peak area
0h
2h
4h
8h
24h
0.1
1.0
10(m
/z)
external transports
time needed forclinical internal
handling / transport
Transportation / processing of blood
Peiyuan Yin
Rainer Lehmann, University Hospital Tübingen, Germany
fold cha
nge of
peak area
0h
2h
4h
8h
24h
0.1
1.0
10(m
/z)
external transports
time needed forclinical internal
handling / transport
Identified metabolitesC2-carnitineC8-carnitineC12-carnitineN-acetylornithineHypoxanthineProline betainePalmitic amideSphingosine 1-phosphateIndoleTryptophanLPC 16:0 LPC 18:0 LPC 18:1LPC C20:3OleamideL-MethionineBiliverdine
Transportation / processing of blood
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Rainer Lehmann, University Hospital Tübingen, Germany
Identified metabolitesC2-carnitineC8-carnitineC12-carnitineN-acetylornithineHypoxanthineProline betainePalmitic amideSphingosine 1-phosphateIndoleTryptophanLPC 16:0LPC 18:0LPC 18:1LPC C20:3OleamideL-MethionineBiliverdine
A targeted metabolomics approach which includedseveral of these metabolites confirmed the findings
B. Kamlage, et al. Clin. Chem. 2014
Transportation / processing of bloodfold cha
nge of
peak area
0h
2h
4h
8h
24h
0.1
1.0
10(m
/z)
external transports
time needed forclinical internal
handling / transport
Rainer Lehmann, University Hospital Tübingen, Germany
New (ongoing) studieGoal:
- define confidence intervals for good and poor quality of bloodsamples in ca. 100 samples (samples were selected at random in a
metabolic ward in our outpatient clinic)
- receive an impression of the number of changed metabolitesmodern, more sensitive mass spec (triple TOF-MS), new analytical strategy
Analytical approaches
non-targeted metabolomics
targeted metabolomics
Transportation / processing of blood
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Rainer Lehmann, University Hospital Tübingen, Germany
positive modedetected ion masses:3137
negative modedetected ion masses:2910
Significant changed metabolite ion masses
unpublished data
0.82 %0.14 %ice water5.09 %1.48 %room temp.
ESI -
0.29 %0.13 %ice water3.6 %0.64 %room temp.
ESI +
4 h2 h114 metabolite ions
148 metabolite ions
n = 30
NON-targeted metabolomics
262 metabolite ion masses were significantly altered
p < 0.05, FDR 0.05
Xinyu Liu
Rainer Lehmann, University Hospital Tübingen, Germany unpublished data
metabolite ion mass A
0h Ice 2h Ice 4h RT 2h RT 4h
n = 30
NON-targeted metabolomics
Potential markers of biobank sample quality
metabolite ion mass D
0h Ice 2h Ice 4h RT 2h RT 4h0
10000
20000
30000
40000
50000
Xinyu Liu
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Rainer Lehmann, University Hospital Tübingen, Germany
76.7 98.3 metabolite ion mass (A)= lactic acid
%-Sensitivity %-Specificity
metabolite ion mass (B) 91.7 98.9
metabolite ion mass (C) 96.7 100
metabolite ion mass (D) 100 100
unpublished data
metabolite (B)metabolite (D)metabolite (C)Lactate
?blood processing delayed by 4 h
NON-targeted metabolomics
n = 30
Rainer Lehmann, University Hospital Tübingen, Germany
blood collection
transportation / processing of blood
storage
repetitive freeze and thaw cycles
Markers of biobank sample quality
biobank
analysis
samplepreparation
dataevaluation /
bioinformaticsvalid results
transport
centri-fugation
plasma / serum
clinicalstudy
bloodcollection
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Rainer Lehmann, University Hospital Tübingen, Germany
Problem: the number of aliquots is limitedleads 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
Biobanking / stored samples
Rainer Lehmann, University Hospital Tübingen, Germany
no significant alterations of metabolite ion masses between fresh controls and 1x frozen plasma samples were detected
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!
unexpectedly, only 4 masses changed significantly after 2x and 4x freeze/thaw cycles!
Biobanking / stored samples
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Rainer Lehmann, University Hospital Tübingen, Germany
and another aspect…. relevant for biobank samples intended for MS-driven –omics analysesAdditives included in the sample collectors may affect the ionization process during an LC-MS run thereby suppressing the ionization of metabolites or introduce interfering compounds.
Planning phase of studies
Rainer Lehmann, University Hospital Tübingen, Germany Yin P, ….Häring H.-U., Lehmann R.: Clin Chem 2013;59:833-45
different S-Monovetten (Sarstedt)
were investigated
consequently, the first important step in the planning and preparation of a study is to check each specific sample collector planned to be used for the generation of biobank samples
Planning phase of studies
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Rainer Lehmann, University Hospital Tübingen, Germany
Summary (1)
Blood collection/storage is the easy part of complex clinical studies,BUTmay heavily affect the quality of biobank samples and consequently the outcome and success of research projects
SOPs
strictly regulate the exposure time of whole blood to room temperature or cooling (as well as the further processing),
BUT
this exposure time may vary from SOP to SOP, e.g. based on logistical problems and resulting compromises in the SOP
and the exposure time may be prolonged in occasional instances
Rainer Lehmann, University Hospital Tübingen, Germany
Summary (2)
The discovered pre-analytical biomarkers may help to identify
a) systematic inaccuracies that arise during processing of whole blood that affect the quality of biobank samples
b) random errors leading to particular outliers in the sample set
and
Investigators should be careful in nominating metabolites identified to be sensitive to preanalytical alterations as biomarker candidates in their research studies
P. Yin, et al. Clin. Chem. 2013 B. Kamlage, et al. Clin. Chem. 2014
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Rainer Lehmann, University Hospital Tübingen, Germany
Summary (3)- Recommendations -
3.) place blood immediately in ice water after drawing until further processing (for a fixed time; ideally not longer than 2 hours)
we prefer (EDTA-)plasma instead of serum as the favorable sample materialprocessing: centrifugation at 4°C, for 10 min, at 2000 × g
5.) handling of biobank samples:- samples should be thawed in ice water- non refrozen plasma aliquots of biobank samples are recommended- mix-up of samples exposed to different freeze and thaw cycles should be avoided
4.) the quality of biobank samples should be tested by using EDTA plasma aliquots before the samples are used in expensive and time consuming analytical projects
1.) the suitability of sample collectors should be tested before startingsample collection
2.) hemolytic samples must be excluded
Rainer Lehmann, University Hospital Tübingen, Germany
Ammonia
Albumin
Alk. Phosph.
Poor sample quality does not necessarily mean that the biobank sample has to be discarded,
but these samples should not be used for high resolution analyses, like metabolomics analysis
Summary (4)- Recommendations -
W. Guder et al., J. Lab. Med. 2002
Stability in whole blood at room temperature Stability in serum / plasma at room temperature
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Rainer Lehmann, University Hospital Tübingen, Germany
Guowang XuXinjie ZhaoPerry YinXinyu LiuShili Chen
Zhongda ZengJia Li
Hans-Ulrich HäringAndreas Fritsche
Norbert StefanRobert Wagner
Erwin Schleicher Andreas Peter
Ann Kathrin PohlHeike Runge
Mareike WalentaMiriam Hoene
Sabine NeukammMagnus Wolf
Christian KlinglerCora Weigert
Dalian Instituteof Chemical Physics
- CAS -
current
funding:NSFC
Bente K. Pedersen Peter Plomgaard
Jakob Hansen Xiaohui LinHai Wei
Dalian University of Technology
Oliver KohlbacherErhan Kenar
Holger FrankenLars Rosenbaum
Andreas Zell
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