Supervised by

Dr. Jawahar Lal

Submitted by
Abhisheak Sharma

Pharmacokinetic & Metabolism Division,

CSIR - Central Drug Research Institute, Lucknow (India)

Dried blood spots (DBS) is a cumulative technique for sample acquisition, transport, archiving, and prospective/ retrospective bioanalysis.

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Number of papers

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0 1970 1980 1990 2000 2010

Year

Results by year till 2012 for searching keyword Dried Blood Spot on PubMed.
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Comparison of DBS method with conventional blood sampling techniques

Blood collection from finger prick / toe prick/ by tail vein Pipetting of blood samples on DBS cards Storage of spotted samples on DBS card with desiccant at room temperature Punching/ cutting of test spots

Drying of DBS card

Analytes extraction by liquid liquid extraction or Solid phase extraction*

Analysis of analytes/ biomarker/ disease

DBS Method V/s

Blood collection from venipuncture

Centrifugation of blood for plasma or serum separation

Storage and transportation of plasma/ serum/ whole blood samples under cold chain

Analytes extraction by liquid-liquid extraction, protein precipitation or solid phase extraction

Analysis of analytes/ biomarker/ disease

Conventional blood sampling techniques

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Selection of Paper

Sample Collection

Drying of Card

Addition of internal standard (IS)

Extraction and analysis

Automation

Calculation and correlation with previous plasma/ 5 serum results

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The DBS cards are composed of a cellulose matrix (filter paper) of specific pore size and thickness. Now-a-days, various commercial DBS cards are available viz., Whatman (GE Healthcare, USA) designed FTA DMPK-A, B, C cards as per type of analysis.
FDA approved DBS cards: Ahlstrom 226-K062932, Whatman 903 and PerkinElmer 226 under 21 CFR 862.1675 as medical device for blood specimen collections.

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Back

 For therapeutic drug monitoring and diagnosis of diseases, whole blood sample is collected from a finger, toe or heel-prick. For PK and TK studies in rat and mouse, blood can be collected via the caudal vein. Although heparin or Ethylenediaminetetraacetic acid (EDTA) can be used as anticoagulant however, EDTA is more suitable than heparin. blood volume for spiking on DBS card depends on the sensitivity of bioanalytical method.
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 DBS cards can be dried for 2 h on a card rack at room temperature or under nitrogen flow. Although drying time of the card will depend upon the type of card as well as sample volume. However, drying of the card is the crucial step for unstable analytes. Various modifications like pH change, temperature and humidity control are recommended for such cases.

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IS can be incorporated before extraction, either directly in extraction tube or in extraction solvent. By pre-spotting on DBS card before addition of blood sample for better results. IS can also be sprayed on DBS card for homogeneity across the spot and reproducibility between the spots. 9

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 Dried cards can be punched out with punching tools. Punched dried card can be used directly (by microfluidics) or by extraction of analytes with suitable extraction solvent.

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After extraction, samples are subjected to analysis. LCMS/MS, desorption electrospray ionization (DESI)-MS, GCMS, MALDI-MS, MALDI TOF-MS, HPLC, isoelectric focusing (IEF)-HPLC, direct laser desorption (LD) TOF-MS, inductively coupled plasma (ICP)-MS, laser ablation (LA) ICP TOF-MS, PCR, ELISA and microfluidics chips have successfully been coupled with DBS method for qualitative and quantitative analysis of blood samples.

An extraction free direct spray ionization technique is reported by Manicke et al. in which analyte is converted into gas phase ions using solvent electrospray by applying a high voltage (3500 V) to the wet substrate.

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 Online automated blood sampling tools (ABS2; Culex) from free moving laboratory animals can be coupled for automated serial sampling (in microliter of blood volume) on DBS cards with high throughput and accuracy.
Pals automated Sample Card And Prep (SCAP) system can be coupled with LC-MS/MS for online drug analysis.

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Calculated plasma concentration =

Analyte concentration in DBS [ 1H +H KRBC/plasma]

If KRBC/plasma or Kblood/plasma is: greater than one, DBS will have higher drug levels than plasma or serum results and DBS will have better PK/PD correlation. equal to one, DBS results should be identical with plasma or serum results and DBS can be an alternate for plasma study. less than one, DBS results should have lower analyte levels than plasma or serum.
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Preclinical pharmacokinetics/toxicokinetic study.

low sampling volumes II. Less number of animal require Offering improved data quality over composite sampling paradigms. Low quantity of new chemical entities (NCE) required during drug discovery experiments. No effect of hemolysis on PK/PD correlation.

Population disease control and clinical pharmacokinetic study.

low sampling volumes II. Cheap Multi-centric clinical investigations Less invasive (Finger/ toe prick v/s venipuncture) Successful in remote areas where transportation facilities, cold chain facility or intravenous sampling may reduce the efficiency of mass policy implementation.
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Therapeutic drug monitoring.

Other applications (Doping, metabolomic profiling, pharmacokinetic drug- drug interaction studies and pharmacogenetics).
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 Can only be coupled with highly sensitive analytical technique. In case of low clearance and saturable blood cell binding of the

compound, blood cell to unbound plasma concentration ratio

will vary with concentration. As a result, whole blood or DBS will not be able to calculate accurate bioavailability as the blood ratios of area under curves will differ. DBS is a not a worth technique for compounds having minimal blood cell uptake (Kblood/plasma 0.55).

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 Dilution integrity is not possible with DBS. Hematocrit, the blood parameter, influences the spread of

blood on DBS card and partially influence the results by

significant inter subject variability. In metabolomic profiling using DBS as some of the

metabolites viz., L-lysine, iminodiacetic acid, DL-threobeta-hydroxyaspartic acid, citric acid, adipamide and adenosine-5-monophosphate, are found to be absent in DBS extracts but are detected in blood or plasma.

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 Using the power of modern analytical technology,

financial and ethical inspiration of DBS method makes

it high-flying technique for blood sampling in a short period of time. Apart from few limitations, DBS is

unquestionably the most ethical and economical

technique for blood sample collection, shipping and storage.

For personalized medication, it may be a boon to the

medical practitioners by getting microbiome and metabolome profiles with single window drug monitoring

and diagnosis of diseases.

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 Modified DBS methods like perforated DBS (PDBS), bilayer dried

plasma spot card or Hemaspot technology should be encouraged to overwhelm the drawback of conventional DBS method. Lots

of advancements are still awaited to resolve the concerns of

repeated or/ additional analyses, homogeneity of spot, hematocrit value for blood, recovery of DBS and reduction of the signal-to-noise ratio by blood cell component separation. Appreciation of regulatory authorities and sponsors is required to use the DBS technology with automation, for expansion of drug discovery in shorter duration with limited resources.
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Thank You

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