symposium 4 biospecimen science · rainer lehmann, university hospital tübingen, germany...

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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


- 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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„….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!


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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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?


....

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



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?


....

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


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Blood collection Transportation Storage

hemolysis delayed processingrepetitive

freeze/thaw cycles

we applied metabolomics to investigated these preanalyticalsteps


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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primary note for the coming slides: Scheme of data evalution

Metabolomics data processing


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)


biobank

analysis

samplepreparation

dataevaluation /

bioinformaticsvalid results

transport

centri-fugation

plasma / serum

clinicalstudy

bloodcollection

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Blood drawing

Hemolysis

n = 10

non-targeted metabolomics

classical parameters

blooddrawing


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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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


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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blood collection

transportation / processing of blood

storage


biobank

analysis

samplepreparation

dataevaluation /


transport

centri-fugation

plasma / serum

clinicalstudy

bloodcollection


© 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“


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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


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

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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


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


Peiyuan Yin


fold cha

nge of

peak area

0h

2h

4h

8h

24h

0.1

1.0

10(m

/z)

external transports



Identified metabolitesC2-carnitineC8-carnitineC12-carnitineN-acetylornithineHypoxanthineProline betainePalmitic amideSphingosine 1-phosphateIndoleTryptophanLPC 16:0 LPC 18:0 LPC 18:1LPC C20:3OleamideL-MethionineBiliverdine


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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




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


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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


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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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


n = 30


blood collection

transportation / processing of blood

storage

repetitive freeze and thaw cycles


biobank

analysis

samplepreparation

dataevaluation /


transport

centri-fugation

plasma / serum

clinicalstudy

bloodcollection

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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


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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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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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


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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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


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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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

symposium 4 biospecimen science · rainer lehmann, university hospital tübingen, germany...

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