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

[email protected]

*: Analytical Chemistry, in press

mailto:[email protected]

mailto:[email protected]


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# of results/year when performing a Pubmed search for "dried blood spots"

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)

*


• 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


"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


Illustration of the hematocrit problem

Ingels et al., Bioanalysis, 2011

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0.58

25-l DBS

Every partial-DBS (punch) based bio-analytical procedure should pre-define a Hct interval in which it can be applied!


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!


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


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


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


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


“…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’?


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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Total [Hb] in function of time

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Obvious Candidate as ‘Hct substitute’: HEMOGLOBIN

(Hb measurement on Cobas 8000)

?


[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!


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


[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 (%)

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Range: 0.19 – 0.63 3 QC’s: Low (0.24), Medium (0.41) & High (0.58)


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


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?


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)


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


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!


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)


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


Y U For your attention

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prediction of the hematocrit of dried blood spots via ... · generation dbs microsampling technique...

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