next generation preanalytics: biomolecular quality and it...

[email protected] Institut für Klinische Chemie Institut für Klinische Chemie

Next Generation Preanalytics: biomolecular

quality and IT approaches

Prof. Dr. Michael Neumaier, MD PhD

Institute for Clinical Chemistry

Medical Faculty Mannheim of Heidelberg University

Mannheim

Germany

[email protected] Institut für Klinische Chemie

Preanalytics, since when a problem ?

„Harnbeschauer aus dem „Augsburger Arzneybüchlein“ (Xylograph from 1502)

from: M. Neumaier, 100 Jahre Klinische Chemie und Naturwissenschaften Mannheim, Selbstverlag (2010)


a problem now !

from: M. Neumaier, 100 Jahre Klinische Chemie und Naturwissenschaften Mannheim, Selbstverlag (2010)


Phases of testing in the medical laboratory

Guder WG et al., Samples: From the Patient to the Laboratory, GIT Publisher (1996)


Blood sample quality, but ...

Lippi G et al., Diagnosis (2018)


Health

from equilibrium to „ex-vivo/in-vitro“ stability


Health Disease



Health Disease specimen



preanalytical stability of medical Biomarkers

Guder WG et al. Die Qualität diagnostischer Proben, Recommendations of DGKL, 2005

WHO/DIL/LAB/991 Rev2 2002


WHO/DIL/LAB/991 Rev2 2002

preanalytical stability of medical Biomarkers

Guder WG et al. Die Qualität diagnostischer Proben, Recommendations of DGKL, 2005


Green RE et al., Science (2010)

The stability of DNA allows for whole genome

analysis from Neandertal bones (40,000 years old)

by NGS with error rates of 0.3 - 5.9%.

Zimmer JS et al., J. Lipid. Res., (2004)

Instability of leucotrienes (40 sec.

in PBS at 4°C); „improved“ to up to 18 min. by FABP proteins in serum.

Range of biomarker stability in biospecimens


the „Elephant-in-the-room“: preanalytics of biomolecular quality


Clinical relevance of molecular decay in clinical specimens

bio

ma

rke

r co

nce

ntr

atio

n

usually unknown

cut-off


usually unknown

bio

ma

rke

r co

nce

ntr

atio

n


cut-off


usually unknown

bio

ma

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atio

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cut-off

2 Variables • sample age • decay


the Y-axis: (Reference „BMB Mk I“ und „BMB Mk II“)

• Blood draw (Serum / Li-Hep-Plasma / EDTA)

– healthy donors (hospital staff through hospital staff physician); (n=128)

– Patients (inflammation, tumour, diabetes); (n=92)

• centrifugation (Mk I)

– Aging the supernatant 1h, 2h, 4h, 8h, 24h at ambient temperature

• no centrifugation (Mk II)

– Aging the whole blood 1h, 2h, 4h, 8h, 24h at ambient temperature

• aliquoting and archiving -20°C, -80°C, liqN2 (stop watch)

• thaw and measure on routine lab equipment


Single parameter analyses (1)

0

1

2

3

4

5

6

7

8

9

1 2 4 6 8

LA

Lact

ate

[mm

ol/

l]

Time [h]

0

10

20

30

40

50

60

70

80

90

1 2 4 6 8

TP

Tota

l Pro

eti

n [g

l/l]

Time [h]

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

1 2 4 6 8

TBIL

Tota

l Bili

rub

in [m

gl/d

l]

Time [h]

0

2

4

6

8

10

12

1 2 4 6 8

CRP

C-r

eac

tive

Pro

tein

[mgl

/l]

Time [h]

Health Disease


RefBMB Mk1 parameters: [Urea]

100.0% Maximum 3,665

99.5% 3,645

97.5% 3,122

90.0% 2,232

75.0% Quartil 1,800

50.0% Median 1,385

25.0% Quartil 1,095

10.0% 0,801

2.5% 0,375

0.5% 0,082

0.0% Minimum 0,000

VISTA imprecision limit: 5%

source Log-weight P-value

Cohort (H/D) 1,949 0,01125 storage temp. 0,829 0,14825


2h

4h

8h

24h

Urea Deviation [+/- %]

RefBMB Mk1/Mk2 parameters: [Urea]


Urea Lactate Homocysteine

RefBMB Mk1/Mk2 parameters: individual changes/stability over time


Stabilities of biomarker from RefBMB and acc. to Guder


HOMOCYSTEINE Serum Ageing LACTATE Serum Ageing

FOLATE Serum Ageing PHOSPHATE Serum Ageing


Factors influencing biomarker stabilities (storage temp., cohort)

Classification: [very unstable (


Profiling using endogenous Decay-Marker

Degradation of exogenous Decay-Marker

Quantitative Determination of biomolecular Quality in biospecimens

Addition of exogenous Decay Marker


RRRRRRR-Ahx-rrrrrrr (RPint)

RRRR-Ahx-rrrrrrr (RP-4; 741 m/z)

Ahx-rrrrrrr (AP; 494 m/z)

2h

24h

4h

8h

exogenous Decay Markers for Protein Quality (Example 2: Quantification using LC-MS/MS)

mod. acc: Findeisen P. et al., Am J Clin Path (2013)


preanalytical assessment of biospecimen quality using exogenous decay marker

Findeisen P et al., Am J Clin Pathol (2013)

Seru

m

Pla

sma


80% classification of sample age by MALDI-TOF

high intermediate low

Fibrinogen a • 1466 [m/z] • 1351 • 1264 • 1207 • 1120 • 905 Complement C4 • 1923 • 1811 • 1723 • 1609 • 1592 • 1482 • 1257 • 1101 • 988 (anchor) Procalcitonin • 1115 • 1002 (anchor)

Serum (n=133) Plasma (n=62)

01h

04h

08h

24h

end.

Compl. C4 PCT

exog.

Compl. C4 PCT

exog.

FGa

e

FGa

Findeisen P et al., CCLM (2018)


Precision of Assessment of Biosample Age (% error in measurement of protein decay by exogenous decay markers)

-100

-50

0

50

100

150

200

0 2 4 6 8 10 Time elapsed [h], verum

Bland-Altman-Plot Exogen

Erro

r [%

]

exogenous


usually unknown

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the X-axis (LabCoder)


LabCoder system

• arm-fixed (both hands-free) LabCoder device (Android; Linux)

• identifies patient (wrist band)

• downloads working list for blood draws from LabCoderAdmin middleware

• assigns tube (UID code; matrix code) according to lab order

• joins patient-ID and tube-ID

• measures fill-level for citrate specimens

• uploads order data to LIS

• middleware customizable to wards´ requirements

• other apps for patient-near services (BP, HR, °C etc.)


LabCoder (evolution)

1st design 1st prototype demonstrator

2nd design 3rd design


LabCoder 3rd Design (CAD, 3D Print)


www.stef-fauser.de (Klackband „Klackinger“ -16€)


LabCoder.App for Android

Test order patient data

ward, rm material quality

code order fill-level

Editing of order


2nd prototype


LabCoder.App for Android (summary)

choose App Labcoder

Clinical

BP /HR (Bluetooth) temperature (Bluetooth) blood sugar

Patient-near services

patient´s appointments Diet/Menu

Commodities

UMM-Navigation


LabCoder IT-surroundings

LIS

LabCoder ADMIN

KIS

LabCoder Ward´s-PC

patient blood draw

ord

ers

Re

sults

do

wn

load

up

load

Laboratory analyzers

DotMatrix ggfs. Rückcodierung

Laboratory


LabCoderADMIN webserver middleware


usually unknown

bio

ma

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atio

n

exact time and slope of decay may allow „retrograde extrapolation“ of test results

cut-off


the Future of Lab Med and the perspectives of Preanalytics

• combining technical PRE with biological PRE

– potential collaboration with biobanking societies

• develop reliable decay characteristics

– time, temperature, health status etc.

• develop reliable procedures

– time stamps

• develop means to assess biological activity in specimens as

surrogate for deterioration (decay markers)

...., but finally ....


the end of the problem? (USS Enterprise, Starfleet AD 2378)

from: Startrek the film, 10 - Nemesis


Thank you for your kind Attention!

[email protected]

next generation preanalytics: biomolecular quality and it...

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