Glycated hemoglobin: a useful post-mortem reference marker in determining diabetes

Glycated hemoglobin (HbA(1c)) has been demonstrated to be a useful marker for long-term glucose control in diabetes. This parameter characterizes each non-enzymatic fixation of glucose on hemoglobin. It is a useful test in addition to periodic glycemia controls since it reflects the mean glycemia of the past 60 days. We studied the conservation of HbA(1c) at 4 degrees C as a function of time with different anti-coagulants and preservatives (3, 6 months, 1 year). A total of 106 tests were performed using the high performance liquid chromatography (HPLC) method dedicated to the semi-automatic analysis of HbA(1c) (Bio-Rad) and we applied the method in forensic cases. Conservation at 4 degrees C was good for as long as 3 months in blood samples collected with fluoride and 6 months in samples collected in a dry or in a heparinized tube. In non-diabetic subjects, HbA(1c) reference values obtained from forensic samples were identical to those of living controls (3.5-6.25% of total hemoglobin). All positive HbA(1c) results were confirmed by a medical evaluation. This method was successfully applied to five forensic cases. In cases of increased acetonemia, acetone or isopropanol are easily measured. However, in some unexplained post-mortem circumstances, increased HbA(1c) permits to differentiate alcoholic or starvation ketoacidosis from the diabetic cases. Glycated hemoglobin should, therefore, be considered the forensic marker of choice in the post-mortem diagnosis of a diabetic disorder and demonstrates its usefulness in post-mortem validation.     

HbA1c as a postmortem tool to identify glycemic control

Estimates suggest that more than 5A million U.S. citizens unknowingly have diabetes and are at increased risk of morbidity and mortality. We evaluated an immunoturbidimetric measurement of glycated hemoglobin (%HbA1c) as a postmortem tool to identify such individuals. Although postmortem samples undergo some degradation, the effects are not sufficient to invalidate the use of the test or method. Using two study populations whose medical history of diabetes was known, we found the mean %HbA1c of the non-diabetics (5.8+/-0.3) to be statistically different from that of the diabetics (12.4+/-2.8). For the population whose disease status was unknown, the %HbA1c ranged from 4.7 to 16.8. For six unknowns whose values exceeded 7.0%. the mean was 11.7%, which did not differ statistically from the diabetic mean (p = 0.6615). These studies suggest that postmortem blood samples can be used to characterize HbA1c values.     

Postmortem pink teeth. Histochemical identification of the causative pigment

The phenomenon of postmortem pink teeth has been reported in subjects who have died suddenly and unnaturally, and whose bodies have been subsequently exposed to a wet or moist environment. Ground and EDTA-decalcified sections of teeth of 21 corpses exhibiting postmortem pink-stained teeth were investigated for the identification of the responsible pigment. With histochemical methods and ultraviolet microscopy, the causative pigment was identified as undegraded hemoglobin. Staining from hemosiderin, bile and bile-related pigments, and porphyrins was ruled out. However, histochemical techniques are unable to reveal the form in which the hemoglobin occurred. The cause of death or time of death had no observable effect on the staining reactions for hemoglobin in the teeth.     

Clinical and forensic examinations of glycemic marker 1,5-anhydroglucitol by means of high performance liquid chromatography tandem mass spectrometry

Postmortem diagnosis of diabetes and a diabetic coma can be difficult because of the lack of characteristic morphological findings. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for reabsorption in the kidneys. Therefore, diabetics with a permanent hyperglycemia show significantly lower serum concentrations of 1,5-AG than non-diabetics. A liquid chromatography-mass spectrometric method for the determination of 1,5-AG in serum and postmortem blood was developed and validated according to international guidelines. Linearity was given between 1μg/ml and 50μg/ml. Recovery rates ranged between 70.8% and 89.8%, the limit of quantification of the procedure was 0.20μg/ml, limit of quantification was 0.55μg/ml. Serum of 199 diabetics and 116 non-diabetics and femoral blood of 31 diabetic and 27 non-diabetic deceased was measured. Average concentrations were significantly (p<0.001) higher in non-diabetics compared to diabetics ante and postmortem. Seven of the diabetics may have died because of a hyperglycemic coma indicated by a sum formula of Traub>450mg/dl. 1,5-AG average concentrations in these deceased were not significantly different to diabetics which did not die because of a diabetic coma. Concentrations of 1,5-AG give a hint for not well controlled diabetes antemortem and postmortem and can be assumed as an additional and alternative information postmortem to the measurement of HbA1c or fructosamine.     

Biochemical measurements of glucose metabolism in relation to cause of death and postmortem effects

This study was performed to examine the relationship between postmortem biochemical values and cause of death. The follow samples were taken from 399 corpses: cerebrospinal fluid (CSF; n = 376, suboccipital), blood (n = 158, femoral vein), and urine (n = 101, at autopsy). (See Table 1 for causes of death) All samples were stored at -80 degrees C. A further 100 samples of blood were later taken and stored at +4 degrees C before testing. Biochemical determinations made were: glucose in CSF, blood, and urine (hexokinase method); lactate (LDH/GPT) and free acetone (HS-gas chromatography) in CSF; hemoglobin A1 in blood (microcolumn technique). In 34 cases fatal diabetic coma was considered verified by morphological and chemical findings. One hundred cases of sudden cardiac death were chosen as the main control group. In 32 of the 34 cases defined above, the value of the formula of Traub (glucose + lactate in CSF) exceeded 415 mg/dl. It is not influenced significantly by hyperglycemia or hyperlactatemia due to factors other than diabetes (i.e., carbon monoxide, asphyxia). After death the value rose till the 30th hpm, then remained stable for at least 1 week. Fatal coma was defined as the ketoacidotic form if free acetone in CSF ranged above 21 mg/l. In these cases, CSF glucose and free acetone correlated positively. Hemoglobin A1 remained stable after death. Its amount was independent from postmortem blood glucose, postmortem interval and total hemoglobin. Furthermore, the manner of storage (-80 degrees or +4 degrees C) had no significant influence on its values. In 29 of 34 cases of fatal coma, Hb A1 exceeded 12.1%. Analysis of urine glucose showed elevated levels (over 500 mg/dl) in diabetic comas. On conclusion, fatal diabetic coma seems indicated as the cause of death if measured values of postmortem biochemistry exceed the following limits: CSF-Traub 415 mg/dl, free acetone (CSF) 21 mg/l; Hb A1 12.1%; urine glucose 500 mg/dl. Most important are the Traub formula and hemoglobin A1. Usually, in fatal coma both values are elevated. If both of them are normal, diabetic coma can nearly be excluded. Combined evaluation of all values is absolutely necessary. Morphology must also always be taken into account. Consequently, a diagnosis of fatal coma can be obtained by a process of elimination.     

抗人血红蛋白胶体金检测试剂条的研制

目的制备法医学检验所用的确定人血的免疫胶体金层析试剂条。方法选取抗人血红蛋白单克隆细胞株,制备其小鼠腹水,从腹水中纯化出单克隆抗体。制备胶体金并用一纯化的单克隆抗体包被,制成免疫胶体金。取玻璃纤维以免疫胶体金浸泡,烘干。在一硝酸纤维素膜上两个不同位置分别点加另一抗人血红蛋白抗体和羊抗鼠IgG。搭建试剂条并检测其灵敏度和特异性。结果制成的免疫胶体金试剂条可对稀释至20万倍的人血红蛋白溶液显示阳性,对法医学检验常见8种动物的血溶液显示阴性。结论所制备抗人血红蛋白胶体金试剂条可以应用于法医学检验。     

Postmortem differential diagnostics of diabetic and alcoholic ketoacidosis

It was shown that ketoacidosis may be an immediate cause of death in subjects suffering chronic alcoholic intoxication. The authors present a list of biochemical studies and parameters that can be used for differential diagnosis between diabetic and alcoholic ketoacidosis.     

Differentiation of fetal and adult bloodstains by pyrolysis-gas-liquid chromatography

A pyrolysis-gas-liquid chromatographic method has been developed for the differentiation of adult and fetal bloodstains. In a blind-coded study, five adult and three fetal bloodstains were correctly identified on the basis of the pyrograms of stain extracts. The differentiation between adult and fetal bloodstains is based on the peak height ratio of two long-retention-time peaks appearing in their pyrograms. The first of these peaks has been tentatively identified as indole derived from the pyrolysis of tryptophan, while the second peak is an as-yet unidentified molecular fragment produced by the pyrolysis of some component of the hemoglobin molecule other than the amino acids tryptophan, tyrosine, and phenylalanine.     

Treatment of paraphilia in young adults with leuprolide acetate: a preliminary case report series

Some juveniles who engage in sexual offenses may have a paraphilia, a psychiatric disorder characterized by a pervasive pattern of deviant and impairing sexual fantasies, thoughts, and/or behaviors. Though there is no known cure for these conditions, paraphilias can be effectively managed using a multimodal treatment approach. This may include the use of psychotherapeutic and pharmacological treatment interventions, including antiandrogen medications. One such agent, leuprolide acetate (leuprolide), a luteinizing hormone-releasing-hormone agonist, has been shown to be effective in reducing paraphilic symptoms in adult patients. To date, however, there is no published data on its use and effectiveness in adolescent and young adult paraphilic patients. This study consists of a case report series of six young adult patients treated with leuprolide. All subjects had been diagnosed with at least one paraphilia (i.e., Pedophilia, Sexual Sadism, Frotteurism, and Paraphilia Not Otherwise Specified). All subjects had been refractory to treatment in a residential program for adolescent sex offenders prior to initiation of leuprolide. All six subjects reported a reduction in sexually deviant symptoms following treatment with leuprolide. Clinicians rated four as much improved and two as moderately improved. The treatment was well tolerated in all six subjects. This preliminary case series supports the conclusion that leuprolide deserves further examination as a potentially safe and effective component in the treatment of young adult patients with paraphilia.     

Accuracy of Identifying Juvenile/Adult Status from Third Molar Development using Prediction Probabilities Derived from Logistic Regression Analysis

The use of third molars in predicting juvenile/adult status (</≥ 18 years) has important legal ramifications. Third molar development was assessed using Köhler's grading on 268 orthopantomograms of Indian subjects. Logistic regression analysis was applied to determine allocation accuracy of juvenile/adult status and the level of probability that is “reliable” in predicting juvenile/adult status. Allocation accuracies ranged between 75.8% and 78.2% for the sexes combined, with minimal male‐female differences. Adults were categorized more accurately than juveniles, suggesting that Köhler's grading puts Indian juveniles at greater risk of unwarranted punishment. In both sexes, juvenile/adult status was “reliably” predicted when the probability was >80% using individual third molars (excepting the lower right third molar in males); combining upper and lower third molars on the left/right sides, “reliable” predictions were possible when the probability was >80% and >90% for females and males, respectively. Overall, “reliable” juvenile/adult status prediction was achieved in c. 36% of subjects.     

Identification of fetal hemoglobin in blood stains by high performance liquid chromatography

A new method for the identification of fetal hemoglobin (Hb F) in blood stains by reverse-phase high performance liquid chromatography is described. Differentiation between fetal and adult blood stains is based on the existence of gamma-chain peaks which are characteristics of Hb F. Very few gamma chains appeared on chromatograms of all the adult blood stains examined. The level of Hb F could be determined by measuring the total of chromatogram gamma-globin chain areas, and expressing it as a percentage of total Hb. Levels in six cord blood stains on filter paper ranged from 81.1% to 91.3% and remained constant for at least 12 weeks. This method is of great value for its simplicity, sensitivity and speed, and most importantly for its reliability in the field of forensic medicine.     

Vitreous Fluid and/or Urine Glucose Concentrations in 1335 Civil Aviation Accident Pilot Fatalities*

Abstract: During aviation accident investigations, vitreous fluid and urine samples from pilot fatalities are analyzed for glucose and blood for hemoglobin A_1c (HbA_1c) to monitor diabetic pilots and to discover other pilots with undiagnosed/unreported diabetes. The prevalence of elevated glucose concentrations in fatally injured pilots was evaluated by searching the Civil Aerospace Medical Institute’s Toxicology Database for the period 1998–2005. Out of 1335 pilots involving 363 vitreous fluid, 365 urine, and 607 vitreous fluid and urine analyses, 43 pilots had elevated glucose in vitreous fluid (>125 mg/dL) and/or in urine (>100 mg/dL). Of the 20 pilots whose blood samples were analyzed, nine had >6% HbA_1c—four were known diabetics, and five were unknown diabetics. Urinary glucose levels were elevated in all 13 known hyperglycemic pilots. A considerable number of pilots (30 of 43) had elevated glucose and HbA_1c (5 of 20), suggesting undiagnosed/unreported diabetic conditions.     

Isotachophoretic analysis of bloodstains: differentiation of human, menstrual, bovine, and ovine bloods

Isotachophoresis, a technique to separate components by constant current electrophoresis, was used to differentiate between bloodstains of male, female, menstrual, bovine, and ovine bloods on cotton cloth and filter paper. Bloodstain analysis by isotachophoresis of stains from male and female subjects showed identical cationic patterns, but gave different profiles in the anionic system. Plasma had one extra peak in the anionic system when compared to the profile of serum. This extra peak is due to the presence of fibrinogen in plasma. Some hemoglobin peaks overlapped with serum protein peaks, but these could be identified by comparisons at lower concentrations. Menstrual blood had a much different pattern than normal human blood as was expected since many more compounds are found in menstrual blood than in normally circulating blood. Human, bovine, and ovine bloodstains showed different profiles both in the cationic and anionic systems. These results indicate that isotachophoresis can be used for the rapid and simple analysis of bloodstains to differentiate reliably human male, female, and menstrual blood and also to distinguish human bloodstains from those of cattle or sheep.     

Postmortem diagnosis of unsuspected diabetes mellitus established by determination of decedent's hemoglobin A1c level.

Although approximately 15.7 million Americans have diabetes mellitus, with the vast majority having type 2 diabetes, it is estimated that as many as 5.4 million are undiagnosed. The present case illustrates that undiagnosed diabetes can be a factor in otherwise unexplained deaths. A 39-year-old white male with no significant past medical history other than alcohol abuse was found deceased at his residence. The manner of death appeared to be natural, but no anatomic cause was found. Toxicological analysis revealed a blood ethanol level of 0.02 g/dL and was negative for drugs of abuse. Analysis of the vitreous fluid revealed a glucose level of 502 mg/dL. The blood glucose level was 499 mg/dL, and the hemoglobin A1c (HbA1c) level was 10.6%. Only trace urine ketones were detected, suggesting that the death was the result of hyperglycemic hyperosmolar non-ketosis (HHNK) from unsuspected diabetes. The postmortem HbA1c value serves as a definitive indicator of prolonged hyperglycemia. In order to aid the interpretation of the clinical data, this case is discussed in conjunction with a similar case of a known diabetic patient.     

Discrimination Between Infant and Adult Bloodstains Using Micro‐Raman Spectroscopy: A Preliminary Study

In the present study, we used micro‐Raman spectroscopy with high‐resolution analysis to discriminate between bloodstains from infants and bloodstains from adults. Raman peaks were detected at 674, 754, 976, 1002, 1105, 1127, 1176, 1248, 1340, 1368, 1390, 1560, and 1611 cm^?1; these peaks were derived from hemoglobin, albumin, and glucose. However, a peak was obtained at 1105 cm^?1, which was assigned to histidine; this peak was observed only for bloodstains from adults. Human adult hemoglobin (HbA) is composed of an α₂β₂ tetramer structure, whereas human fetal hemoglobin (HbF) is composed of an α₂γ₂. Therefore, the lack of a Raman peak at 1105 cm^?1 in bloodstains from infants indicates the possibility of two histidine substitutions (His116Ile and His143Ser) in the γ chain of HbF. This study discriminates between bloodstains from infants and bloodstains from adults using micro‐Raman spectroscopy, with beneficial implications in forensic science.     

The effects of adult and peer social initiations on the social behavior of withdrawn and aggressive maltreated preschool children

The effects of programmed peer- and adult-mediated play initiations were compared for socially dysfunctional, maltreated preschool children. An N = 1, alternating treatments design was used to compare these strategies. Two withdrawn nonmaltreated (control), two withdrawn maltreated, and three aggressive maltreated children served as subjects. Dyadic play sessions were videotaped and rated at the completion of the study by naive raters according to an 18-category observation system. Overall, the control children showed no difference in levels of interaction between peer and adult conditions. The withdrawn maltreated children showed a clear preference for interaction with the peer over the adult. The aggressive maltreated children showed a clear preference for interaction with the peer over the adult. The aggressive maltreated children showed a clear preference for interaction with the adult. They also demonstrated a decrease of positive behavior and increase in negative behavior in response to the programmed initiations. Implications of these differential responses were discussed.     

On the possibility of postmortem diagnosis of diabetes mellitus

The present study has demonstrated that elevated level of glycated haemoglobin in cadaveric blood may be regarded as an absolute diagnostic criterion for diabetes mellitus irrespective of other macro- and/or micropathomorphological signs of this disease.     

CHILD ABUSE,NEGLECT, AND VIOLENT CRIMINAL BEHAVIOR*

Using a prospective cohorts design, official criminal histories for a large sample of substantiated and validated cases of physical and sexual abuse and neglect from the years 1967 through 1971 (n = 908) were compared with those of a matched control group (n = 667) of individuals with no official record of abuse or neglect. Abused and neglected subjects had higher rates of having an adult criminal record than controls and a larger number of arrests as an adult. Based on a logit analysis, a model using four explanatory variables (age, sex, race, and abuse/neglect status) provided a good fit. In comparison with controls, abused and neglected subjects also had a higher frequency of arrests for violent offenses as adults; however, this was due primarily to significantly more adult violent offenses by abused males. Support for the cycle of violence is discussed as well as sex differences in the results, limitations of the findings, and implications for further research.     

Phenolphthalein False‐Positive Reactions from Legume Root Nodules

Presumptive tests for blood play a critical role in the examination of physical evidence and in the determination of subsequent analysis. The catalytic power of hemoglobin allows colorimetric reactions employing phenolphthalein (Kastle‐Meyer test) to indicate “whether” blood is present. Consequently, DNA profiles extracted from phenolphthalein‐positive stains are presumed to be from blood on the evidentiary item and can lead to the identification of “whose” blood is present. Crushed nodules from a variety of legumes yielded phenolphthalein false‐positive reactions that were indistinguishable from true bloodstains both in color quality and in developmental time frame. Clothing and other materials stained by nodules also yielded phenolphthalein false‐positive reactivity for several years after nodule exposure. Nodules from leguminous plants contain a protein (leghemoglobin) which is structurally and functionally similar to hemoglobin. Testing of purified leghemoglobin confirmed this protein as a source of phenolphthalein reactivity. A scenario is presented showing how the presence of leghemoglobin from nodule staining can mislead investigators.