Quantitative assessment of the percent fat in domestic animal bone marrow

Measurement of the amount of fat in femoral bone marrow can provide a quantitative assessment of the nutritional status of an individual animal. An analytical method is presented for quantitating the percent fat in bone marrow from three domestic species: bovine, canine, and equine. In this procedure, fat is extracted from bone marrow using pentane, and the percent fat recovered is determined gravimetrically. Based on analyses from adult animals (normal body condition scores), the average percentage of fat in the bone marrow was >80%. In cases in which animals have been diagnosed as emaciated or exhibit serous atrophy of fat (body scores of 1 or 2), the femoral bone marrow fat was less than 20%. In domestic animals, bone marrow fat analysis can be a useful, quantitative measure that, when used in conjunction with all other data available, can support a diagnosis of starvation or malnutrition.     

Determination of opiates in postmortem bone and bone marrow.

Bone and bone marrow of a fatally poisoned heroin addict were analyzed by FPIA and GC-FID, immediately after death. A piece of the bone from the above case was buried for 1 year and analyzed by the same procedure. Morphine was detected in all specimens at concentrations of 195, 340 and 155 ng/g for bone marrow, bone and buried bone, respectively. A loss of 54.4% of morphine concentration was observed during 1-year burial. Such findings have potential forensic value in cases of skeletonized remains.     

Colonization of diatoms in and on porcine bone substrate and considerations of diatom ecology for forensic science

The presence of diatom algae in bone marrow has been used as forensic evidence of drowning for several decades; however, these studies are based on known or suspected recent drowning events. This study addresses the potential for diatoms to enter the bone marrow of skeletal remains, that is, de-fleshed long bones post-mortem. In laboratory and field experiments, bones were either inflicted with two access points by a cut and acid pitting or left intact. The bones were submerged in water for at least 1 week and up to 3 months. Samples of the bone surface and marrow were inspected for diatoms. The analysis considered the time required for diatoms to enter marrow and whether genus characteristics like size or mobility affect entry. The presence of an access point influenced diatom entry in that bones without an introduced access point had zero to one diatom present in the marrow, whereas a bone with an access point had >150 diatoms present in the marrow. The results of both laboratory and field phases suggest that diatoms will reliably colonize bone in as quickly as 1 week, establishing and maintaining communities for at least 3 months. However, the bone surface assemblages differ from the source community. Bone marrow displayed even more restrictive access to diatom colonization, resulting in communities dominated by small raphid diatoms. Based on these findings, we suggest some caveats on the use of diatoms as trace evidence in forensic science with recommendations for future avenues of research.     

Morphine and 6-acetylmorphine concentrations in blood, brain, spinal cord, bone marrow and bone after lethal acute or chronic diacetylmorphine administration to mice

The aim of this study was to evaluate postmortem incorporation of opiates in bone and bone marrow after diacetylmorphine (heroin) administration to mice. Mice were given acute (lethal dose of 300 mg/kg) or chronic (10 and 20 mg/kg/24 h for 20 days) intraperitoneal administration of diacetylmorphine. The two metabolites of diacetylmorphine, 6-acetylmorphine (6-AM) and morphine, were extracted from whole blood, brain, spinal cord, bone marrow and bone (after hydrolysis) using a liquid/liquid method. Quantification was performed by gas chromatography-mass spectrometry (GC/MS). Results showed that after acute administration, opiates were present in all studied tissues. Morphine concentrations appeared to be higher than those of 6-AM in blood (52.4 microg/mL versus 27.7 microg/mL, n=12), bone marrow (87.8 ng/mg versus 8.9 ng/mg, n=6) and bone (0.85 ng/mg versus 0.43 ng/mg, n=6), but 6-AM concentrations were higher than those of morphine in brain (14.0 ng/mg versus 7.4 ng/mg, n=12) and spinal cord (27.8 ng/mg versus 20.8 ng/mg, n=12). No correlation was found for both compounds between blood concentrations and either brain, spinal cord, bone or bone marrow concentrations while a significant one was found between brain and spinal cord concentrations either for morphine (r=0.89, n=12, p<0.001) or 6-AM (r=0.93, n=12, p<0.001), the concentration being higher in spinal cord than in brain. When bones were stored for 2 months, only 6-AM remained in bone marrow but not in bone. After chronic administration, mice being sacrificed by cervical dislocation 24 h after the last injection, no opiate was detected in any studied tissues. Further studies are required, in particular in human bones, but these results seem to show that 6-AM could be detect in bone marrow several weeks after the death and could be an alternative tissue for forensic toxicologist to detect a fatal diacetylmorphine overdose, even if no correlation between blood and bone marrow was observed. On the other hand, neither bone tissue nor bone marrow will allow the confirmation of a chronic diacetylmorphine use.     

A comparative study of ethchlorvynol levels in blood versus bone marrow

Bone marrow may be utilized as an alternative biological sample in cases where uncontaminated blood samples are not available for analyses. Bone marrow/blood ratios of ethchlorvynol as a function of time and dosage level were determined in 40 rabbits. A modified quantitative analysis that produced accurate and reproducible results was employed for the determination of ethchlorvynol levels. Further, ten blood and bone marrow samples containing ethchlorvynol were chosen to study the effects of storage for a period of 24 hours. Studies of blood and bone marrow ethchlorvynol levels with time showed no linear relationship. Bone marrow/blood ratios as a function of dose resulted in close mean averages with a wide range of values. Significant losses in both blood and bone marrow ethchlorvynol levels were evidenced in most of the samples subjected to the 24-hour storage study.     

Determination of morphine in postmortem rabbit bone marrow and comparison with blood morphine concentrations

The aim of this study is to predict how long after time of death a buried body could be analyzed for opiates in soft tissues and to show the accessibility and suitability of bone marrow as a useful toxicological specimen from buried bodies. Morphine solutions were injected in nine albino rabbits. Doses ranged from 0.3 to 1.1 mg/kg with 0.1 mg/kg increments. One hour after the injections, the rabbits were sacrificed. Blood, urine and bone marrow samples were collected for analysis. After the whole bodies were buried, femur bone marrow specimens were collected on the seventh and fourteenth days. CEDIA was used to monitor morphine contents of the collected samples. All experimental cases showed that the increase in the given morphine doses correlated with the increase in blood and bone marrow morphine concentrations. High morphine concentrations were detected in urine samples, but there was no correlation between the urine and blood or urine and bone marrow morphine concentrations. Statistically meaningful increases in bone marrow morphine concentrations were found parallel to increase of blood morphine concentrations. Seventh and fourteenth day postmortem morphine concentrations also followed this correlation. Morphine concentrations in bone marrow at 7 and 14 day postmortem decreased consistently when compared with bone marrow morphine concentrations collected immediately after death. We conclude that in sudden death when other specimens are unavailable due to degradation, bone marrow can be a most useful specimen. Further experimental research in this area is required to validate bone marrow as an alternative tissue.     

Maternal death due to non-traumatic fat embolism

The aim of this report is to document a case of non-traumatic fat embolism (NTFE) and to address the need for considerition of fat embolism in suspicious deaths resulting from respiratory distress in the postpartum period. A 28-years-old woman autopsied at the Morgue Department of the Council of Forensic Medicine is included to the study. This female became unconscious and developed respiratory distress 4 h after delivery, and this was followed by respiratory arrest. External examination revealed resuscitation marks and normal postmortem changes. Light microscopy revealed massive fat embolization involving most of the alveolar capillaries on several sections. Only in one particular area was a bone marrow embolus. Pathological diagnosis of the lung was diffuse pulmonary fat embolism. There was no evidence of other organ involvement with emboli. Other visceral organs showed no striking findings other than mild congestion. The cause of death was considered to be respiratory insufficiency resulting from severe fat embolism of the lungs.     

Plasma versus bone marrow desipramine: a comparative study

Correlation between plasma and bone marrow tricyclic antidepressants has not been studied before. Two groups of rabbits were given 10 and 20 mg of desipramine/kg body weight, respectively. Desipramine was administered to the animals once daily by mouth for 5 days. On the fifth day the animals were sacrificed and blood and bone marrow samples were collected and analyzed using a high performance liquid chromatographic (HPLC) method. Data showed that a correlation exists between bone marrow and blood desipramine. The bone marrow desipramine concentration increased as its blood levels increased. The average ratio of bone marrow to blood desipramine +/- S.D. (standard deviation) in both dosage groups was 37.2 +/- 4.46 with a range of 30.99-44.82. This investigation is promising and shows that bone marrow could be used as an alternative tissue in the absence of a suitable blood sample.     

Blood versus bone marrow methanol concentrations in rabbits

Postmortem methanol levels in bone marrow and heart blood were determined in rabbits. The average ratio of heart blood concentration to observed bone marrow concentration in 36 rabbits was 2.6 ± 0.6 with a range of 1.5 to 4.2. Correcting for the lipid content of the bone marrow decreased the average ratio, reduced the ratio range and improved the correlation. The heart blood to corrected bone marrow ratio was 1.6 ± 0.3 with a range of 1.2 to 2.9. Direct injection gas chromatographic techniques were employed to quantitate methanol concentrations.     

Comparative study of ethanol levels in blood versus bone marrow,vitreous humor,bile and urine

Post-mortem ethanol levels in blood were compared to corresponding levels in rib bone marrow, vitreous humor, urine and bile. In forensic toxicology, a good correlation between blood and a tissue or body fluid is needed to estimate a blood alcohol concentration when blood is unavailable or contaminated. In this study, direct injection and headspace gas-chromatographic techniques were employed to quantitate the ethanol concentrations. Comparable findings by these two techniques showed a reproducibility of results. When the determined bone marrow ethanol levels were corrected for the lipid fraction, a consistent correlation could be established between ethanol levels in blood and bone marrow. The relationship (linearity and ratio range) between ethanol levels in blood and corrected levels in bone marrow was better than that between blood and vitreous humor, bile or urine. This study showed that blood ethanol levels can be predicted by extrapolating the corrected rib bone marrow ethanol level.     

Plasma versus bone marrow flurazepam concentration in rabbits

Methods for the quantitation of flurazepam in plasma and bone marrow were developed for the purpose of determining the relationship between flurazepam concentrations in both tissues.Albino New Zealand rabbits, given flurazepam in doses of 5, 10, or 20 mg/kg, were sacrificed either one or three hours after drug administration. Flurazepam concentrations in plasma and bone marrow were determined utilizing a gas-liquid chromatograph equipped with an electron capture detector. The average plasma/bone marrow flurazepam ratios were 0.033 ± 0.012 and 0.024 ± 0.012 for rabbits sacrificed one hour and three hours after dosing, respectively. This study showed that a range of plasma flurazepam levels can be estimated from known bone marrow concentrations. The overall mean plasma/bone marrow ratio for all rabbits used in this study was 0.029 ± 0.012 with a range of 0.010 to 0.055.     

Evaluation of post-mortem ethanol concentrations in pericardial fluid and bone marrow aspirate

This study confirmed post-mortem ethanol concentrations in pericardial fluid and bone marrow aspirate in comparison with those in the blood in medicolegal autopsy cases (n = 140, within 48 h post-mortem). The specimens were examined by head-space gas chromatography/mass spectrometry. Ethanol concentrations in the pericardial fluid (y) were approximately equivalent to those in peripheral blood (x): y = 0.99x + 0.02, n = 44, r = 0.972. A high stomach ethanol concentration (>10 mg/ml) appeared to mildly affect the pericardial levels. There was no significant interference in drowning cases. Ethanol concentrations in bone marrow aspirates (y) also showed a good correlation with those in the peripheral blood (x): y = 0.77 x + 0.02, n = 20, r = 0.981. A dissociation was observed in cases of delayed death from hemorrhagic/traumatic shock and elderly victims. These findings suggest that pericardial fluid and bone marrow aspirate can be used as an alternative material when adequate blood specimens are not available.     

Blood, bone marrow and eye fluid ethanol concentrations in putrefied rabbits

The effect of putrefaction on postmortem blood, bone marrow and eye fluid ethanol levels was evaluated in rabbits. Control and dosed animals were sacrificed and stored at either room temperature (approx. 19 degrees C) or cold temperature (approx. 3.5 degrees C) for as long as 28 days. Control animals stored at room temperature showed ethanol levels in the bone marrow that peaked at 7 days after sacrifice, followed by decreases to a nondetectable level at 21 days. Overall decreases were demonstrated in bone marrow of dosed rabbits stored at room temperature for all postmortem intervals. The control animals stored at low temperature showed no ethanol in the bone marrow and blood until 21 days after sacrifice. Dosed rabbits stored at low temperature showed no significant changes in blood and marrow ethanol until 21 days after sacrifice.     

Behavior of genetic markers in recipients after bone marrow transplantation and problems in forensic medicine

The authors report studies on four pairs of donors and recipients in bone marrow transplantation (BMT). A broad range of gene markers at 41 gene loci, including 11 red blood cell markers, 5 human lymphocyte antigen (HLA) types, 12 serum protein markers, 5 red cell enzyme markers, and 8 salivary markers were evaluated before and after BMT over 2 months. As a result, 9 out of 41 gene loci of genetic markers in recipients were transformed into the donor type. BMT between family members may lead to transformation of gene markers, but within a pattern compatible with family inheritance patterns, and no genetic paradox will be found in later surveys of familial genetic relationships. However, in a personal identification system in forensic medicine using genetic markers as an index, the appearance of a phenotype incompatible with a blood relationship is possible after BMT with a non-blood-relative donor. This result is similar to the inheritance pattern observed after artificial insemination by a donor's semen (AID), a more complete out-of-family cross.     

中国汉族成人肱骨近端CR片特征与年龄的相关性

目的探讨中国汉族成人肱骨近端CR片的特征随年龄变化的规律,以推断年龄。方法 210例中国汉族成人肩关节CR片,其中男性124例,年龄范围18~83周岁,女性86例,年龄范围18~60周岁,选取与年龄变化关系密切的肱骨骨髓腔高度、肱骨近端骨骺骨小梁变化、肱骨大结节内骨小梁变化、锁骨骨髓腔肩峰侧骨小梁变化4项指标,观察不同年龄各指标的变化进行统计分析。结果四项指标均是影响年龄变化的重要因素。肱骨近端骨骺骨小梁变化、锁骨骨髓腔肩峰侧骨小梁变化对年龄的预测影响较大,肱骨骨髓腔高度、肱骨大结节内骨小梁变化稍小。利用以上指标建立的回归方程能很好的反映成人肱骨的增龄性变化,预测骨龄较为准确。结论利用成人肱骨骨髓腔高度、肱骨近端骨骺骨小梁变化、肱骨大结节内骨小梁变化、锁骨骨髓腔肩峰侧骨小梁变化可推断年龄。     

Utility of immunoassay in drug screening in skeletal tissues: sampling considerations in detection of ketamine exposure in femoral bone and bone marrow following acute administration using ELISA

Detection of ketamine exposure in skeletal tissues by automated enzyme-linked immunosorbent assay (ELISA) and gas chromatography with electron capture detection (GC-ECD) is described. Rats (n = 18) received 0, 15, 30, or 75 mg/kg ketamine hydrochloride acutely (i.p.), and were euthanized within 15 min or 1 h. Ketamine was extracted from ground femoral bone by methanolic incubation followed by liquid-liquid extraction (LLE), while marrow was homogenized in alkaline solution, and then underwent LLE. Extracts were analyzed by ELISA, and subsequently by GC-ECD following derivatization with trifluoroacetic acid anhydride. The effect of tissue type (i.e., diaphyseal bone vs. epiphyseal bone vs. bone marrow) on the immunoassay response was examined through determination of binary classification test sensitivity (S) and measurement of the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. The %DA varied significantly between different tissues examined under a given dose condition, and generally decreased in the order marrow > epiphyseal bone > diaphyseal bone, at all dose levels examined. Measured S values for marrow, epiphyseal bone, and diaphyseal bone were 100%, 77%, and 23%, respectively (75 mg/kg dose). These results suggest that the type of skeletal tissue sampled and position sampled within a given bone (diaphyses vs. epiphyses) are important parameters in drug screening of skeletal tissues.     

Autolytic changes in blood cells and other tissue cells of human cadavers. I. Viability and ion studies

The viability of white blood cells, spermatozoa of the epididymis, cells from minced spleen, lymph node and lung, and cells aspirated from the bone marrow of the sternum was studied using the vital dye exclusion test. The material comprised 123 medicolegal autopsy cadavers which had been stored in a mortuary cold room at +4 °C up to 10 days after death. Cells which excluded trypan blue were found in various specimens from all cadavers. However, there was marked individual variation in the results. The loss of viability of the white blood cells showed a moderate correlation (r = ?0.78) with the increase in the post-mortem (p.m.) time, whereas the results for other tissues were not so significant. The K⁺ and Mg²⁺ and haemoglobin content and the osmotic resistance of the red cells correlated poorly with the p.m. time. The present results suggest that despite the general assumption that autolytic changes proceed rapidly at the cellular level, individual cells and especially blood cells may remain viable for a long time in cadavers kept at +4 °C.     

Widespread myocardial and pulmonary bone marrow embolism following cardiac massage.

Widespread fatal massive bone marrow embolism of the coronary and the pulmonary arteries are described in a 71-year-old man with a respiratory disease. Cardiac massage was carried out during the cardiac arrest. In autopsy findings there were no fractures of the ribs or the sternum. The authors suggested that the cardiac massage was the most important predisposing factor for the bone marrow embolism.     

Blood Versus bone marrow ethanol concentrations in rabbits and humans

Post-mortem ethanol levels in bone marrow and heart blood were determined in rabbits and humans. The average ratio of blood/femur marrow ethanol in 39 rabbits was 2.10 ± 0.23 with a range of 1.27 – 3.50. In 18 human autopsy cases the average ratio of blood/rib marrow ethanol levels was 2.16 ± 0.32 with a range of 1.65 – 2.94. The average blood/marrow ratios in humans and rabbits were comparable. However, a wider range of ratios was noticed in the rabbit study.