Hargrove, Veronica M. PhD, FTS-ABFT; Molina, D. Kimberley MD
From the Bexar County Medical Examiner’s Office, San Antonio, TX.
Manuscript received May 16, 2013; accepted July 2, 2013.
The authors report no conflicts of interest.
Reprints: Veronica M. Hargrove, MS, Bexar County Medical Examiner’s Office, 7337 Louis Pasteur Dr, San Antonio, TX 78229. E-mail: email@example.com.
It is known that postmortem drug concentrations can vary depending on the sampling site and that, in general, central sites have higher drug concentrations than do peripheral sites. It has also been suggested that clamping the femoral vessel before drawing the sample may eliminate possible contribution from central sites. Morphine is a commonly prescribed and commonly encountered opiate medication that is often found in postmortem examinations, both as a cause of death and also as an incidental finding. It is important to understand the degree of postmortem redistribution of morphine to peripheral sites and whether clamping the femoral vessel can eliminate postmortem redistribution of morphine to ensure the correct interpretation of postmortem morphine concentrations. Morphine drug concentrations were evaluated in clamped and unclamped femoral vein blood samples at 3 different times before autopsy, and no significant change in either the clamped or the unclamped femoral vein morphine concentration was seen over time. Furthermore, no significant difference was found between the clamped and unclamped blood concentrations at any period. Therefore, it can be concluded that for morphine, unclamped femoral blood samples do not show significant redistribution from central sites within the first 24 hours after death in bodies kept refrigerated at 4°C.
In most medical examiner (ME) offices, bodies are transported to the office and refrigerated upon arrival before examination, which may occur 24 hours or even longer after receipt of the body. Postmortem redistribution is a well described phenomenon in postmortem toxicology, and it has been shown to occur within the first 24 hours after death. Previous studies have collected samples upon arrival to the mortuary or field samples to limit the effects of this process.1–3 However, this procedure can also cause issues with the examination and could potentially result in loss or contamination of evidence.
Although the issue of postmortem redistribution is important for most drugs, it is of particular importance for opioids/opiates, in which the concentrations can vary greatly between patients with chronic pain and opiate-naive patients because of tolerance. Morphine, in particular, is a commonly prescribed and commonly encountered opiate medication found in postmortem examinations. In addition, whether and how much morphine redistributes to femoral vessels after death are unknown. It has long been accepted that drugs with a volume of distribution greater than 3 to 4 L/kg are expected to redistribute postmortem because of their tissue-concentrating effect.4 Morphine’s volume of distribution is 2 to 5 L/kg, which means that this drug falls at the cutoff for whether it should redistribute postmortem. Animal studies with rat models have shown that there is indeed an increase in postmortem heart blood morphine concentrations over time,5,6 although a more recent study with swine showed that total morphine concentrations did not change over time or between sampling sites.7 Human studies regarding morphine have also been conflicting. Several articles have shown that there is a redistribution of morphine postmortem,1,8,9 whereas Logan and Smirnow10 reported that in 32 morphine-related deaths in which there were two separate femoral blood collections, there was no significant redistribution of morphine. Gerostamoulos and Drummer2 reported that in 21 heroin-related deaths in which there were a matched admission and an autopsy femoral sample, the median concentrations of total morphine and free morphine were similar in both samples.
In response to the issue of postmortem redistribution, some toxicologists advise to clamp the femoral vessel before drawing blood samples to prevent contamination of peripheral blood from higher drug concentrations in central blood.1,11 This clamping is thought to prevent blood from being inadvertently pulled from more central sites, such as the vena cava, thereby preventing the mixing of higher central drug concentrations with the lower peripheral drug concentrations. Although this issue was examined and refuted in a previous study,12 morphine, in particular, was not included.
The present study was designed to assess whether it is necessary to obtain blood specimens for morphine testing as soon as possible to prevent contamination by postmortem redistribution or whether the current procedure of placing bodies in a cold (refrigerated) environment is sufficient to prevent a significant amount of redistribution from occurring before autopsy and, therefore, sample collection. In addition, we also sought to address whether the femoral vein requires clamping before blood is drawn to prevent contamination from more central sites (ie, inferior vena cava) creating falsely elevated peripheral blood concentrations.
MATERIALS AND METHODS
Cases examined at the Bexar County Medical Examiner’s Office between February 2011 and February 2012 that had a history of known morphine use were selected for this study. Cases involving children and homicides and cases with extensive medical intervention and/or significant blood loss or trauma were excluded. Upon receipt of the body at the morgue, an incision was made down both femoral regions, and the left and right femoral veins were exposed. One of the veins was then clamped, and the other vein was left patent. Blood was then collected (<3 mL) from both veins at 3 different times before autopsy—at its initial receipt at the morgue and then at intervals of approximately 6 hours until the examination was performed. Blood samples were collected in gray top tubes (sodium fluoride preservative) and stored at −20°C until assayed. The bodies were kept refrigerated at 4°C upon arrival at the morgue and throughout the extent of the sampling. Morphine analysis was accomplished using a solid phase extraction procedure, and samples were analyzed using liquid chromatography-tandem mass spectrometry. Statistical analysis was performed using multivariate analysis of variance to assess any change in the morphine concentrations over time and between the clamped and unclamped veins.
A total of 18 cases met the inclusion criteria for this study. The mean age of the decedents was 80 years, with a range from 52 to 96 years. Most of the cases were men (55.6%), with women accounting for 44.4%.
Clamped and unclamped femoral blood morphine concentrations were measured at 3 different times within 24 hours after death. The mean time before the first draw was 5 hours (range, 3–9) after death, the mean time for the second draw was 12 hours (range, 8–14) after death, and the mean time for the third draw was 21 hours (range, 15–24) after death.
The mean morphine concentrations at the first, second, and third draw times in the clamped vein were 0.171 mg/L, 0.170 mg/L, and 0.155 mg/L, respectively, ranging between 0.020 and 0.597 mg/L. The median concentrations for the clamped vein were 0.095 mg/L, 0.108 mg/L, and 0.097 mg/L. Figure 1 shows a graph of the mean concentrations from the first draw to the third draw in the clamped vein. The mean morphine concentrations at the first, second, and third draw times in the unclamped vein were 0.162 mg/L, 0.174 mg/L, and 0.168 mg/L, respectively, ranging between 0.019 and 0.600 mg/L. The median concentrations for the unclamped veins were 0.086 mg/L, 0.092 mg/L, and 0.088 mg/L. Figure 2 shows a graph of the mean concentrations from the first draw to the third draw in the unclamped vein.
Statistical analysis found that there was not a significant change in the morphine concentration in either the clamped or the unclamped femoral vein over time. In addition, analysis also showed that there was no significant difference between the clamped and unclamped femoral vein concentrations at any period. Subclavian blood concentrations were also available for the third draw time, and statistical analysis (paired Student t test) showed that at this time point, there is a statistically significant difference between subclavian blood and femoral blood (P=0.036), with the subclavian concentrations being greater than the femoral blood concentrations (mean subclavian blood concentration, 0.366 mg/L).
Our study shows that morphine does not exhibit significant postmortem redistribution to the femoral vessels when bodies are kept refrigerated at 4°C. This was also the finding shown by Logan and Smirnow10 when they examined morphine-related deaths with two separate femoral blood collections. In their study, a cutdown incision was also done, but no clamping of the vessel followed and bodies were also kept at 4°C. In Logan and Smirnow’s10 study, the time between death and first collection ranged from 3 to 144 hours (mean, 17.3 hours), whereas the interval from first collection to autopsy ranged from 3 to 43 hours (mean, 17.4 hours). It could be argued that with long postmortem intervals, morphine could have redistributed before the first sampling. However, our study has a much shorter postmortem interval with all 3 samples, including the unclamped samples, being obtained before 24 hours postmortem. In addition, we show that morphine concentrations are stable in the femoral vein for the first 24 hours after death. Logan and Smirnow10 also reported consistently higher ventricular blood concentrations when compared with femoral or iliac blood. Our study also supports this finding because subclavian blood was consistently higher than femoral blood. This further enforces the idea that femoral blood is the ideal sample when interpreting toxicology, and blood from central sites should be interpreted with caution.
As mentioned earlier, it has been suggested that clamping the femoral vessels before sampling may eliminate possible contribution from central sites.1,11 This study also showed that there was also no significant difference between morphine concentrations in the clamped femoral and unclamped femoral vessels. A previous publication has also come to this conclusion for other drugs.12 In this study, less than 3 mL of blood was drawn at every time point; therefore, it can be concluded that for volumes up to 9 mL, clamped and unclamped femoral blood will show similar morphine blood concentrations.
In summary, the current procedures followed by most ME offices in the United States of placing bodies in a refrigerated environment before examination, with blood samples being obtained at the time of examination, seem to be sufficient to prevent significant peripheral postmortem redistribution of morphine. In addition, it is not necessary to clamp the femoral vein of such bodies to prevent central contamination of the specimens. However, although the current study shows this to be true for morphine, more research needs to be done for other drugs, specifically those with higher volume of distribution, because refrigeration may not be enough to prevent postmortem redistribution to femoral vessels and clamping of the femoral veins may be necessary. As always, forensic toxicologists and MEs alike should review their toxicology results on a case-by-case basis, considering all the variables involved before rendering a decision regarding cause and manner of death.
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