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A Preliminary Investigation into Retrospective Calculation of In-Vivo Drug Concentrations in Dried Crime Scene Blood. Dwain C. Fuller*, Patricia Pisana, Veterans Affairs North Texas Health Care System, Dallas, TX, U.S.A. Why?.

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slide1

A Preliminary Investigation into Retrospective Calculation of In-Vivo Drug Concentrations in Dried Crime Scene Blood.

Dwain C. Fuller*, Patricia Pisana, Veterans Affairs North Texas Health Care System, Dallas, TX, U.S.A.

slide2
Why?
  • Evaluating injured drivers who may have fled the scene of an accident.
  • Evaluating whether a wounded aggressor who has fled the scene was possibly drug-impaired.
  • Because law enforcement asks.
analytical obstacles
Analytical Obstacles
  • What was the original blood volume?
  • Are drugs stable in dried blood?
  • Can one adequately recover drugs from dried blood?
approach
Approach
  • Phase 1
    • Determine if the original volume of blood can be calculated from the weight of its dried residue.
  • Phase 2
    • Determine the stability and recovery of some representative drugs from a dried blood matrix.
phase 1 weight volume ratio
Phase 1 – Weight/Volume Ratio
  • Selected 50 random blood specimens submitted for CBC analysis.
    • CBC’s selected to avoid any particular disease state
    • CBC’s are submitted in 5 mL purple-top tubes containing an average of 7.4 mg EDTA as an anticoagulant, but no preservative
    • Each tube contained 3.5 - 4 mL blood
phase 1 weight volume ratio1
Phase 1 – Weight/Volume Ratio
  • Demographics
    • Gender: Males – 48, Females – 2
    • Age: Mean – 63 (33-90)
    • Race:
      • Caucasian – 28
      • African American – 13
      • Hispanic – 3
      • Unknown - 6
phase 1 weight volume ratio2
Phase 1 – Weight/Volume Ratio
  • Specimens were mixed for at least 15 min.
  • Weighing boat weighed and weight recorded.
  • 500 µL of blood was pipetted into weighing boat.
  • Total weight was immediately recorded.
  • Boat was dried for at least 72 hours at room conditions (71 ±1⁰F, Relative humidity: 42 ±8%)
phase 1 weight volume ratio3
Phase 1 – Weight/Volume Ratio
  • Boat with dried blood was re-weighed and weight recorded.
  • Weight of dried blood per milliliter of liquid blood was calculated.
other considerations
Other considerations
  • Weight of dried blood was corrected for weight of EDTA anticoagulant (2 mg/mL).
  • One analyst performed all pipetting to avoid interindividual differences in technique.
  • A few specimens were subjected to a 60⁰C oven for an additional 24 hours and re-weighed to determine if drying conditions were adequate.
results
Results

Mean: g dried blood/mL liquid blood = 0.215± 0.025 g (0.161 - 0.259)

CV = 11.6 %

Prediction Uncertainty: approximately ± 23% with 95% confidence

also calculated
Also calculated:
  • Blood density
  • % Water
comparison to other studies
Comparison to other studies

Density: 1.052 ± 0.020 g/mL Present Study1.055 g/mL (Karch, Forensic Issues in Alcohol Testing, CRC Press, 2007)1.06 g/mL (Cutnell, et al. Physics, 4th edition, Wiley, 1998)1.025 – 1.125 g/mL (Benson, Katherine, MCAT Review, Emory University,1999)1.043 – 1.057 g/mL

(Hinghofer-Szalkay, et al., Continous Monitoring of blood volume changes in Humans, Journal of Applied Physiology, Vol 63, 1987)

comparison to other studies1
Comparison to other studies

Percent water:

79.6 ± 2.4 % g/g Present Study

76.9 – 82.0 % g/g 80.5 % g/g – Females 78.9 % g/g – Males

Lijnema, et al., Gravimetric determination of the water concentration in whole blood, plasma and erythrocytes and correlations with hematological and clinicochemical parameters. ClinicaChimicaActa, 214, 1993

limitations of study
Limitations of study
  • Sample size
  • Age
  • Gender
  • Race
  • Disease states (diabetes, etc.)
  • EDTA blood may not be representative of crime scene blood
phase 2 drug stability and recovery
Phase 2 – Drug Stability and Recovery
  • Pooled EDTA blood and screened for drugs of interest
  • Spiked blood with amphetamine, methamphetamine, MDA, MDMA, codeine, morphine, hydrocodone, hydromorphone, cocaine, ecgonine methyl ester, and benzoylecgonine
  • Determined original concentration
phase 2 drug stability and recovery1
Phase 2 – Drug Stability and Recovery
  • Prepared 1 mL aliquots in weighing boats and dried as previously described
  • Analyzed for target drugs as convenient over a period of weeks and months
  • Compared results of dried specimens converted the concentration to the original liquid blood concentration
brief analytical method
Brief Analytical Method
  • Dried blood specimen was transferred into a disposable 16 x 100 mm culture tube.
  • Dried blood specimen was ground to a powder using a 10 mm diameter glass stir rod.
  • Powdered dried blood was transferred to a tared tube for analysis and weight was recorded.
brief analytical method1
Brief Analytical Method
  • Reconstitute dried blood with buffer used for extraction either liquid:liquid or SPE.
  • Add appropriate internal standard(s).
  • Rotate on laboratory rotator for 10 minutes.
  • Shake vigorously, (centrifuge if proceeding with SPE extraction.)
  • Proceed with extraction as normal.
recovery compared to original spiked blood
Recovery(Compared to original spiked blood)

*These values may reflect some breakdown of cocaine to EME

cocaine studies
Cocaine studies

Because of expected lability of cocaine and its metabolites, their stability was determined by comparison to a freshly spiked liquid blood specimen using the original spiking solution stored at -80⁰C, rather than the original liquid blood specimen itself.

observations on cocaine stability in dried blood
Observations on cocaine stability in dried blood

Benzoylecgonine is considerably more stable in dried blood at room temperature than is EME.

Isenschmid, Levine, Caplan. JAT, 13:250; 1989

observations on cocaine stability in dried blood1
Observations on cocaine stability in dried blood

Benzoylecgonine probably breaks down to ecgonine. Thus ecgonine would probably be an important target analyte to demonstrate cocaine use when analyzing dried blood.

Logan. JAT, 25:219; 2001

Skopp, Klingman, et al. Ther. Drug. Monit. 23:174; 2001

a special note about cocaine stability in dried blood
A special note about cocaine stability in dried blood.
  • Present study
    • Loss of 44% of cocaine in dried blood in 14 days
  • Alfazil and Anderson, JAT, 32:511; 2008
    • Loss of 19.9% of cocaine in dried blood spots on filter paper in one month at room temperature
    • blood matrix was prepared by diluting packed cells with isotonic saline
a special note about cocaine stability in dried blood cont
A special note about cocaine stability in dried blood. (cont.)
  • Baselt, Journal of Chromatography, 268, 1983, 502-505
    • Cocaine loss in unpreserved plasma was more rapid than that of unpreserved whole blood
    • This may reflective of a higher pseudocholinesterase concentration in plasma compared to whole blood.
  • Thus Alfazil and Anderson’s results may be skewed due to their choice of matrix.
observations of cocaine stability in dried blood vs liquid blood
Observations of Cocaine stability in dried blood vs. liquid blood
  • Both cocaine and BE appear to be more stable in unpreserved dried blood than liquid blood.
  • Cocaine appears to degrade more to BE rather than EME in unpreserved dried blood as compared to liquid blood.
limitations of study1
Limitations of Study
  • Spiked EDTA blood may not be representative of crime scene blood
    • Clotting
    • Protein binding
    • Issues regarding removal dried blood from differing substrates
  • Actual crime scene blood may be subjected to a wide array of temperatures and weather conditions
future studies
Future studies
  • Evaluate larger more diverse population
  • Evaluate cocaine and metabolite concentration changes in the short term
  • Evaluate more drugs
  • Evaluate authentic blood specimens
    • Logistically difficult
    • Postmortem specimens at autopsy?
  • ??????
conclusions
Conclusions

It may be possible to quantitate drugs in unpreserved dried blood obtained from a crime scene, convert the drug concentrations to their previous liquid blood equivalent, and accurately assess the uncertainty of this process.