dried blood spots

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Submitted by Abhisheak Sharma Dried Blood Spots: Concepts, Present Status and Future Perspectives in Bioanalysis (BIO-CDRI-4-002) Supervised by Dr. Jawahar Lal Pharmacokinetic & Metabolism Division, CSIR - Central Drug Research Institute, Lucknow (India)

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An analytical technique for bioanalysis.

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Dried Blood Spots: Concepts, Present Status and Future Perspectives in Bioanalysis (BIO-CDRI-4-002)

Submitted byAbhisheak Sharma Dried Blood Spots: Concepts, Present Status and Future Perspectives in Bioanalysis(BIO-CDRI-4-002)

Supervised byDr. Jawahar LalPharmacokinetic & Metabolism Division,CSIR - Central Drug Research Institute, Lucknow (India)

1Dried blood spots (DBS) is a cumulative technique for sample acquisition, transport, archiving, and prospective/ retrospective bioanalysis.

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Results by year till 2012 for searching keyword Dried Blood Spot on PubMed.

10/11/20133DBS MethodConventional blood sampling techniques

Comparison of DBS method with conventional blood sampling techniques

V/s

10/11/20134Tools and Techniques10/11/20135Selection of PaperThe 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.Treated v/s UntreatedBack10/11/20136proprietary reagents - lyse cells, denature proteins, inactivate enzymes and pathogens, and prevent the growth of bacteria.

Example: DMPK A&B cards

untreated: no impregnated chemicals to interfere with the analysis.Example: DMPK C and FTA cards

Inhouse cards: Citric acid/ TCA/ by Guowen et al. used citric acid solution on DBS card to stabilize their drug candidate (KAI-9803) from thiol-disulfide exchangeSample CollectionFor 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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Drying of CardDBS 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.

10/11/20138Addition of internal standard (IS)

10/11/20139Extraction and AnalysisDried 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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10/11/201311After extraction, samples are subjected to analysis. LC-MS/MS, desorption electrospray ionization (DESI)-MS, GC-MS, 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.

AutomationOnline 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.

10/11/201312Calculation and correlation with previous plasma/ serum resultsIf 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.13DPSApplications of DBS for investigation of drugsApplications of DBS for investigation of biomarker or diseases10/11/201314DBS Applications and Advantages10/11/20131510/11/201316Limitations of DBS technique

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).

1710/11/201318Dilution 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-threo-beta-hydroxyaspartic acid, citric acid, adipamide and adenosine-5-monophosphate, are found to be absent in DBS extracts but are detected in blood or plasma.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.

Conclusion10/11/201319

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. 10/11/201320Modified 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.Thank You

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