1. DBS – Process & Review GHANSHYAM

2. 3Rs No need to explain…………… GOAL IS ANIMAL SAVING R1 REPLACEMENT R2 REFINEMENT R3 REDUCTION

3. What is DBS? DBS is a technology used since decades but only in the last five years it been considered for routine blood analysis. It was first introduced by Guthrie et al. in 1963 in the context of neonatal screening of phenylketonurea Dried Blood Spot (DBS) is a technology that can be used to determine drug concentration in PK/TK studies from whole blood.

4. Guideline Support ICH S3A says …. “The quantification of systemic exposure provides an assessment of the burden on the test species and assists in the interpretation of similarities and differences in toxicity across species, dose groups and sexes. The exposure might be represented by plasma (serum or blood) concentrations or the AUCs of parent compound and/or metabolite(s).” ….…The choice of analyte and the matrix to be assayed (biological fluids or tissue) should be stated and possible interference by endogenous components in each type of sample (from each species) should be investigated. Plasma, serum or whole blood are normally the matrices of choice for toxicokinetic studies”

5. Advantages of DBS Smaller blood volume required Less sample processing No centrifugation No storage/transfer at frozen temperature Less animals required 30% less rat – no separate TK animals are required 60% less mice in serial blood collection in TK Less API, Man-power, Food etc. Stress due to blood loss can be reduced V d can be managed well.

6. Blood Collection and Sampling Blood sampling by different routes, by different tools is been tested by different researchers. Typically, routine sampling techniques should be adapted and validated for NCE’s.

7. Blood Collection and Sampling Aliquot 15μL blood per spot 3 spots per sample plus ‘spare’ Using a pipette or capillary

8. Drying Dry for approx 2 hours at room temperature Humidity level may interfere in drying – May take longer duration Ready for ship and/or store in sealable bags containing desiccant

9. If Shipping Required Samples classified as “non-hazardous” for shipping The dried filter papers can be shipped at ambient conditions with a simple envelope and in a plastic pouch and preferably with some desiccant material. CDC (Centre for Disease Control and Prevention) "Guidelines for the Shipment of Dried Blood Spot Specimens" state that dried spots can be transported via normal postal systems without special mailing cartons.

10. Punching of Cards Analytical sample obtained by punching small circular disc (typically 3 mm) from centre of DBS Manually Automation – BSD1000

11. Sample Processing Extract disc in organic solvent (typically methanol) containing internal standard Quantitation by validated LC-MS/MS assay DBS system from Prolab GmbH

12. Acetaminophen in Human DBS – On Card Stability Data of Human DBS on FTA paper stored at room temperature for 113 days. N= 6 replicates

13. Acetaminophen in Human DBS – 10-fold Sample Dilution 10,000 ng/mL sample extracted from card then diluted 10-fold using extract from blank DBS samples (n=6)

16.  No immediate storage is required which will help to preserve the quality of the sample collected.  Issues in plasma drug stability can be resolved

17.  Long term studies  (9 animals/sex/dose group in TK) can be avoided  MNT studies  (6 animals/timepoint/dose group/sex) can be sampled more precisely, more samples per animal

18.  Additionally Main Toxicity animals can be used to collect the blood samples for TK as only 15 µl blood will be required instead of 250 µl)

19.  Help for better correlation of HP data with drug exposure as same animals’ data is available for analysis.

20.  Repeat analysis is possible from same DBS card.

21.  Cost Saving by less API, less food, cage change, man hours etc.

22.  Juvenile Toxicity Studies

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