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Dried Blood Spot – Use in Animal Experimentation

12 May

WHAT IS IT?

 In a simple language, the Dried Blood Spot (DBS) technology is a technology in which blood (whole) spot is placed on a specially manufactured blotting paper and after drying, it can be used for further analysis in disease diagnosis, pharmacokinetics, and toxicokinetics in clinical as well as in pre-clinical trials.

 

WHAT IS 3Rs?

In publication of The Principles of Humane Experimental Technique, the Russell and Burch (1959) have coined the concept of 3Rs.

Three Rs includes (1) Replacement, (2) Reduction, and (3) Refinement.

For more information, please visit the BioSim homepage at http://www.biosim-network.net

 

BRIEF HISTORY OF DBS

 The first use of DBS analysis was described by Bang in 1913 for the estimation of blood glucose. Later, the use and application of DBS was well defined by Dr. Robert Guthrie in 1963 for the screening of neonatal inherited metabolic disease, phenylketonuria.

 

USE OF DBS IN PRE-CLINICAL RESEARCH

 I have observed that researchers are sometimes hesitating to adapt the new technologies possibly because of few mentioned reasons.

1)      lacking of published research data.

2)      used to current technology.

3)      Unknown FEAR.

ADVANTAGES OF DBS

 Guideline Support

 In ICH S3A it is mentioned that….

“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 toxicokinetics studies”

Lesser blood volume required (15µl per sample).

Approximately 15µl per spot required which is very less.  Total four spots will not take more than 60 µl of whole blood, which is very less compare to 250 µl of blood for routine analysis. Only one spot is sufficient enough for analysis. Four spots are for backup and reanalysis.

 

No sample processing.

 As this sample collection procedure is direct spot type and no sample processing required which is opposite to conventional system in which centrifugation is required and plasma separation and storage of it within typical time frame is mandatory.


No special storage condition required (-70 °C).

In conventional system, centrifuged plasma sample needs to be stored as soon as possible at deep freezing condition. In DBS, no special storage condition is required. The spotted blood sample can be dried at room temperature (avoid excess humidity level) for around 2 hours and then it can be stored at room temperature (!) with sufficient desiccant material.


Easy transportation (can be transported at ambient temperature with proper desiccant).

If transportation to extra institutions is required then the DBS cards with proper and enough desiccant material can be transported easily using simple envelop at ambient temperature condition.

Additionally, CDC (Centre for Disease Control and Prevention) “Guidelines for the Shipment of Dried Blood Spot Specimens” has stated that dried spots can be transported by normal postal systems without special mailing cartons.

 

Fewer animals required

 I will tell you how…

1) In rat studies, satellite group may not be needed as main toxicity group of animals can be used for toxicokinetics sampling as very less amount of blood is required.

2) In mice studies, like MNT or Repeated dose studies, no sparse sampling is required, or we can reduce the animal usage by taking blood 4 to 5 times from one animal instead of conventionally two samples per animals. (Supports 3Rs)

 

Stress due to blood loss can be reduced (as less blood volume required).

Without any explanation, every researcher will agree on this. Focus on changes in PK/TK parameters due to excess blood loss.

Few more “hidden” advantages.

1) If we measure exposure levels from main toxicity animals instead of separate TK group animals (conventional), it will help to correlate the exposure data with histopathology, hematology, biochemistry, organ weight, any additional unexpected toxicity in few animals etc.

2) Less animal usage supports 3Rs. If you compare sparse sampling, drug exposure data versus single animal drug exposure data, definitely, data derived from single animal will make you feel comfort.

3) DBS can be useful in Juvenile toxicity study.

4) Combine all the advantages and imagine “cost saving” – your boss will be happiest person in this world.

5) Few scientists have worked very extensively in this technology. They have concluded that this technology has good reproducibility, sample dilution support (up to 10X), sample storage stability (113 days at ambient temperature (!) Wow), better compound and metabolite stability, negligible intra and inter run variability, no effect on temperature at the time of collection,

6) Effect on drug plasma concentration due to thawing of plasma sample can be avoided.

 

PROCEDURE

Procedure is very simple to perform. See the image shown below.

 

Procedure for DBS

Procedure for DBS



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2 Comments

Posted by on May 12, 2011 in Toxicology

 

2 responses to “Dried Blood Spot – Use in Animal Experimentation

  1. Dr . Amit

    May 16, 2011 at 4:44 pm

    Thanks for providing this valuable information, that is really very much helpful to us.

    One more thing I would like to know from you is that..I need guidance on parasites of laboratory animals and how to identify them or the treatment for the same.

     
    • ghanshyamvaghasiya

      May 16, 2011 at 7:32 pm

      Dear Dr. Amit

      Please give me time of one week. I will definitely provide you details on this

      Happy Learning

      Ghanshyam

       

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