Prediction of the hematocrit of dried blood spots via ... · generation DBS microsampling technique...
Transcript of Prediction of the hematocrit of dried blood spots via ... · generation DBS microsampling technique...
Stove Christophe EBF, November 15th, 2012
Prediction of the hematocrit of
dried blood spots via potassium
measurement on a routine clinical
chemistry analyzer*
Sara CAPIAU, Veronique STOVE, Willy LAMBERT, Christophe STOVE
Laboratory of Toxicology, Ghent University, Belgium
*: Analytical Chemistry, in press
Stove Christophe EBF, November 15th, 2012
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DBS analysis: expanding like never before
→ Bio-analytical applications in preclinical development, toxicology, TDM, …
Analytes (applications): • DNA, RNA (genetics); e.g. genotyping, … • Proteins
protein (enzyme) activity analysis antibody-based analysis
• Small molecules endogenous (e.g. metabolite profiling)
exogenous (drugs & toxicants)
*
Stove Christophe EBF, November 15th, 2012
• Overall easy sampling
(self-sampling possible)
• Minimally invasive
• Only small volume required
• Representative matrix (blood)
• In most cases: stabilizing effect
(long-term at roomt°)
• Convenient storage & transport
• Straightforward extraction & analysis
• Economic (↓ solvents, …)(↓ animals)
• Can relatively easy be automated
• Only small volume available
sensitive analysis required
• Risk of contamination
should be controlled for by e.g.
incurred re-analysis
• Extensive validation required
cfr. additional parameters: volume/
punching site / hematocrit(!)
• In postmortem work: volumetric
application
(≠ spreading of lysed blood)
• Capillary [ ] ≈ Venous [ ] ??
• Issue of Internal Standards
The general pros and cons of DBS sampling
Stove Christophe EBF, November 15th, 2012
• Overall easy sampling
(self-sampling possible)
• Minimally invasive
• Only small volume required
• Representative matrix (blood)
• In most cases: stabilizing effect
(long-term at roomt°)
• Convenient storage & transport
• Straightforward extraction & analysis
• Economic (↓ solvents, …)(↓ animals)
• Can relatively easy be automated
• Only small volume available
sensitive analysis required
• Risk of contamination
should be controlled for by e.g.
incurred re-analysis
• Extensive validation required
cfr. additional parameters: volume/
punching site / hematocrit(!)
• In postmortem work: volumetric
application
(≠ spreading of lysed blood)
• Capillary [ ] ≈ Venous [ ] ??
• Issue of Internal Standards
The general pros and cons of DBS sampling
Stove Christophe EBF, November 15th, 2012
"Current thinking is that this issue
(hematocrit) needs to be addressed
before practical application of
DBS analysis can progress to the
next level, and any direct analysis
technique needs to be compatible
with this solution.“ Abu-Rabie, Bioanalysis, 2011
“Hematocrit is currently identified as the single
most important parameter influencing the
spread of blood on DBS cards, which could
impact the validity of the results generated by
DBS methods, affecting the spot formation, spot
size, drying time, homogeneity and, ultimately, the
robustness and reproducibility of the assays.” Timmerman et al., Bioanalysis, 2011
“Hematocrit effect is clearly a
major hurdle to the success of
any DBS method and attempting
to ignore or avoid it is not an
option in a regulated environment.” Fan & Lee, Bioanalysis, 2012
“In summary we found that most metabolites used
for newborn screening depend on hematocrit and
on position of the disk. The effects of hematocrit
and position of the disk were sometimes
additive and sometimes even synergistic.” Holub et al., Clinica Chimica Acta, 2006
“…approach could be to normalize the measured concentrations in the punched
disc to another endogenous component of blood that also varies with hematocrit.” Denniff & Spooner, Bioanalysis, 2010
The hematocrit problem in literature
Stove Christophe EBF, November 15th, 2012
Illustration of the hematocrit problem
Ingels et al., Bioanalysis, 2011
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25-l DBS
Every partial-DBS (punch) based bio-analytical procedure should pre-define a Hct interval in which it can be applied!
Stove Christophe EBF, November 15th, 2012
Proposed ‘solutions’ for the hematocrit problem (1)
Avoid sampling bias: use complete DBS rather than DBS punches
Meesters et al., Bioanalysis, 2012
Youhnovski et al., Rapid Comm. Mass Spectrom., 2011
Pre-cut dried blood spot (PCDBS): an alternative to dried blood spot (DBS) technique to overcome hematocrit impact
Dried matrix on paper disks: the next generation DBS microsampling technique for manging the hematocrit effect in DBS analysis
Li et al., Bioanalysis, 2011
Perforated dried blood spots: a novel format for accurate microsampling
→ All require volumetric application of DBS!
Stove Christophe EBF, November 15th, 2012
Calculate Hct: Measure DBS area of volumetrically applied DBS
Denniff & Spooner, Bioanalysis, 2010
The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs
Proposed ‘solutions’ for the hematocrit problem (2)
V= 3 *h * (d2+d*b+b2) Tomtec, 2012
Calculation of frustrum volume
→ Also requires volumetric application of DBS!
→ All volumetric approaches are difficult to sustain (or simply not feasible) when aiming at patient self-sampling (e.g. at home)!
Use of DBS area to derive Hct
Stove Christophe EBF, November 15th, 2012
Li et al., Rapid Comm. Mass Spectrom., 2012
The use of a membrane filtration device to form dried plasma spots for the quantitative determination of guanfacine in whole blood
Proposed ‘solutions’ for the hematocrit problem (3)
Use plasma instead of blood: Generate DPS instead of DBS
Barfield & Wheller, Analytical Chemistry, 2011
Use of dried plasma spots in the determination of pharmacokinetics in clinical studies: validation of a quantitative bioanalytical method
→ “classical” plasma preparation needed
→ promising, more studies needed
Stove Christophe EBF, November 15th, 2012
“…approach could be to normalize the measured concentrations in the punched
disc to another endogenous component of blood that also varies with hematocrit.” Denniff & Spooner, Bioanalysis, 2010
A ‘hematocrit substitute’ should:
• correlate with Hct (i.e. # RBC’s)
• be stable
• be easy to measure in 3-mm punches
• be universal
• show minimal inter-individual (non-Hct-related) variation
What are the requirements for a ‘hematocrit substitute’?
Stove Christophe EBF, November 15th, 2012
Orsini et al., Clinica Chimica Acta, 2010
Semi-quantitative method for determination of hematocrit in DBS, using data collected in HPLC hemoglobin variant testing
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Obvious Candidate as ‘Hct substitute’: HEMOGLOBIN
(Hb measurement on Cobas 8000)
?
Stove Christophe EBF, November 15th, 2012
[K+] as ‘Hct substitute’?
• K is present mainly intracellularly (intracell. [K+] >> plasma [K+])
• K is expected to be stable
• [K+] easily measurable in 3-mm DBS punches?
• K is universal
• [K+] are tightly controlled no large inter-individual variation is expected
RBC are the main cellullar constituent of blood (>99% of cells)
Bring a 3-mm DBS punch in a small tube
Add 50 µl 2.5 mM KCl in ultrapure H2O
Spin down Shake 5’ (@1400 rpm) @ RT
Spin down
(1) Transfer 40 µl
Measure [K+] in 90 µl via indirect potentiometry using the ISE (Ion Selective Electrode) module of
the Cobas 8000 Clinical Chemistry Analyzer
(2) Transfer 50 µl
YES!
Stove Christophe EBF, November 15th, 2012
Does [K+] correlate with Hct?
5 calibration lines: DBS prepared on Whatman 903 paper from blood with increasing Hct
R2=0.9939
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• Linear • Homoscedastic
Stove Christophe EBF, November 15th, 2012
[K+] predicts Hct: validation
On 4 different days: 2 calibration lines (DONOR 1): DBS prepared on Whatman 903 paper from blood with ↑ Hct
Range: 0.19 – 0.63
>99.5% of hospital population
Relative frequency of Hct in a hospital setting (Ghent University Hospital, 1-year-data)
Hct (%)
Rela
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Stove Christophe EBF, November 15th, 2012
[K+] predicts Hct: validation
On 4 different days: 2 calibration lines (DONOR 1): DBS prepared on Whatman 903 paper from blood with ↑ Hct
Range: 0.19 – 0.63 3 QC’s: Low (0.24), Medium (0.41) & High (0.58)
Stove Christophe EBF, November 15th, 2012
[K+] predicts Hct: validation
On 4 different days: 2 calibration lines (DONOR 2): DBS prepared on Whatman 903 paper from blood with ↑ Hct
Range: 0.19 – 0.63 3 QC’s: Low (0.24), Medium (0.41) & High (0.58)
Stove Christophe EBF, November 15th, 2012
Influence of storage on [K+]
[K+] is not affected by: - short-term storage of DBS at elevated t° - long-term storage at room t°
Extract can be stored for at least 3d @ 4°C
20h
Stove Christophe EBF, November 15th, 2012
Mild volume effect, which, however, does not exceed 15%
No effect of punch location on [K+]
Influence of DBS volume and punch location on [K+]
Owing to a in saturation?
Stove Christophe EBF, November 15th, 2012
Application of the developed procedure on patient samples (n = 111)
After correction for the neg. bias: LoA’s of ± 0.049
Comparison of calculated Hct (from DBS from Li-heparin blood) with measured Hct (K2-EDTA blood; Sysmex analyser)
Stove Christophe EBF, November 15th, 2012
Slope: 1.036 [0.943 – 1.141]
Intercept: - 0.012 [-0.042 – 0.018]
Slope: 0.938 [0.861 – 1.015]
Intercept: 0.019 [-0.004 – 0.042]
Application of the developed procedure on patient samples (n = 111)
(after bias correction)
Very good correlation between
Calculated Hct &
Measured Hct
Stove Christophe EBF, November 15th, 2012
Evaluation of assay reproducibility
49 samples were reanalysed 7 days after the original analysis
2/3 of the data points should lie between ± 20%
Fulfilled!
Stove Christophe EBF, November 15th, 2012
Conclusions
• An easy and straightforward protocol for K-extraction from 3-mm DBS punches has been set up
• Evaluation of the [K+]-concentration, using a routine clinical chemistry analyzer, allows prediction of the approximate Hct
• The procedure has been validated in terms of: • Accuracy & precision • Stability pre- & post-extraction • Influence of punch location & volume spotted
• The procedure has been applied on DBS from patient samples, demonstrating:
• Practical applicability • Assay reproducibility (via incurred sample reanalysis)
Stove Christophe EBF, November 15th, 2012
Future Perspectives
• Apply procedure on real capillary DBS (requires measurement of true capillary Hct!)
• Evaluate whether automated extraction & punching can further narrow down the LoA’s possibly even more accurate prediction of Hct
• Evaluate whether Hct prediction effectively overcomes the Hct problem for a given DBS-based analytical procedure
Stove Christophe EBF, November 15th, 2012
Y U For your attention
Thanks to …