Palliative pain therapy at the end of life and forensic medicine issues.

An 83-year-old woman with a history of Alzheimer's disease and breast cancer died at home while receiving palliative pain therapy with oral morphine from her family for metastatic breast cancer. Allegations of mistreatment were made, and this case was ultimately referred to the Office of the Chief Medical Examiner, State of Maryland. An autopsy failed to identify any injuries or residual cancer, leaving no anatomic explanation for the pain that had been presumed to be metastatic breast carcinoma involving bone. The blood free morphine concentration was 5,200 ng/ml, and the total morphine concentration was 15,000 ng/ml. This case demonstrates the challenges and difficulties in forensic medicine when faced with the interpretation of toxicologic results at the end of life.

Lethal combination of tramadol and multiple drugs affecting serotonin.

The death of a 36-year-old alcoholic man who died after developing seizure activity while being treated with tramadol, as well as with venlafaxine, trazodone, and quetiapine, all of which interact with the neurotransmitter serotonin, is reported. The decedent, who had a history of chronic back pain, alcoholism, depression, mild hypertensive cardiovascular disease, and gastritis, had just been discharged from the hospital after 4 days of alcohol detoxification treatment. During the admission, no withdrawal seizures were noted. The morning after discharge, a witness observed the decedent exhibiting seizure activity and then collapsing. An autopsy was performed approximately 6 hours after death, and the anatomic findings were consistent with seizure activity and collapse, which included biting injuries of the tongue and soft-tissue injuries of the face. Toxicologic analysis identified tramadol, venlafaxine, promethazine, and acetaminophen in the urine; tramadol (0.70 mg/L) and venlafaxine (0.30 mg/L) in the heart blood, and 0.10 mg of tramadol in 40 ml of submitted stomach contents. No metabolites, such as acetate, acetone, lactate, and pyruvate, were found in the specimens that would be characteristically found in a person with alcohol withdrawal syndrome. The threshold for seizures is lowered by tramadol. In addition, the risk for seizure is enhanced by the concomitant use of tramadol with selective serotonin reuptake inhibitors or neuroleptics, and its use in patients with a recognized risk for seizures, i.e., alcohol withdrawal. The cause of death in this individual was seizure activity complicating therapy for back pain, depression, and alcohol withdrawal syndrome. The data in Adverse Event Reporting System of the Food and Drug Administration from November 1, 1997 to September 8, 1999 was reviewed along with a MEDLINE search from 1966 to the present. This case appears to be the first reported death caused by seizure activity in a patient taking tramadol in combination with drugs that affect serotonin.

Ferrous sulfate toxicity: a review of autopsy findings.

Ferrous sulfate is the leading cause of accidental pediatric poisonings. Despite the requirement for child-resistant packaging for any oral iron product with 250 mg or more per container, the incidence has continued to increase. Although the clinical presentation of iron toxicity has been well described, pathologic findings in human tissue and correlation with clinical data are scant. We reviewed autopsies from the Armed Forces Institute of Pathology of 11 children who died from ferrous sulfate toxicity. Clinical data, morphologic changes, and iron levels in tissue were evaluated. The children's ages ranged from 11 to 36 mo. Prominent iron deposition in gastric and small intestinal mucosa was associated with necrosis, with some cases demonstrating prominent vascular iron deposition. The clinical courses were rapid and progressed from Stage I to Stage III. These observations were correlated with increased levels of iron in various tissues, as determined by analytical atomic absorption spectrophotometry. The morphologic and chemical analysis data provide information on the pathogenesis of ferrous sulfate poisoning; the vascular iron deposition may be related to subsequent hemorrhage. In the liver the periportal necrosis is probably a direct cytopathic effect of the highest levels of iron carried to these cells by the portal blood flow.

Renal tubular dysgenesis in twin-twin transfusion syndrome.

In twin-twin transfusion syndrome (TTTS), the disparity in circulation is reflected in discordant fetal growth, urine output, and amniotic fluid accumulation. The effect of uneven shunting of the growth factor and nutrient-rich vasculature on development and differentiation of the kidney has not been well studied. We analyzed renal tubular growth and differentiation in 25 fetal autopsies with TTTS (13 donors and 12 recipients, including 9 sibling pairs) between 18 and 33 weeks gestation. Immunohistochemical markers for fumarylacetoacetate hydrolase (FAH), Leu-M1, and Lotus tetragonolobus (LTA) were used to identify proximal convoluted tubules, and epithelial membrane antigen (EMA) was used to demonstrate distal convoluted and collecting tubules. FAH appeared to be more specific and reliable than either Leu-M1 or LTA in the identification of proximal tubules. Donors tended to demonstrate a paucity of proximal tubules with crowding of glomeruli characteristic of renal tubular dysgenesis (RTD). The degree of dysgenesis was greater in later gestations and associated with more severe growth restriction. Donors in TTTS are at risk for the development of RTD. Several authors suggest ischemia as the underlying cause of "acquired" RTD. However, in this setting there is no evidence of cell death or necrosis, and we suggest that hypoperfusion leading to decreased glomerular filtration is the underlying etiology, with the severity of RTD related to the degree of shunting.

Characterization of acetaminophen: molecular microanalysis with Raman microprobe spectroscopy.

The in situ spectroscopic identification of acetaminophen in a fatal overdose case is described. Numerous techniques have been used to analyze acetaminophen in biological fluids, however, the use of nondestructive spectroscopic techniques has not been documented. In this investigation, the demonstration of the drug material was established by using the laser Raman microprobe technique, providing an accurate identification by virtue of the drug's molecular fingerprint characteristics. Material found on the deceased was collected and placed on metal (aluminum-coated) plated slides and excited with the 514.5 nm line of an argon ion laser, which was focused to a 1 micron spot size using a high-resolution optical microscope. Spectra of acetaminophen particles with an average size of 5 to 8 microns were obtained. The Raman spectrum of this drug contains characteristic group frequencies assigned to the C = O at 1649 cm-1, the N-H deformation mode at 1620 to 1612 cm-1, the bendstretch mode of the H-N-C = O at 1562 cm-1, the C-H bending mode at 1325 cm-1, and the phenyl ring stretch at 799 cm-1, respectively. The results reported here demonstrate the capability of laser Raman microprobe as a useful adjunct tool for the identification of foreign materials in forensic pathology.

An analytical comparison of cobalt cardiomyopathy and idiopathic dilated cardiomyopathy.

Toxic cardiomyopathy (TC) has a rapid clinical course and morphologically resembles idiopathic dilated cardiomyopathy (IDC). To further characterize TC, we used light microscopy to compare lesions caused by cobalt (Co) to those of IDC. Cobalt levels were also measured as a chemical marker to differentiate TC from IDC. We reviewed cases with TC and IDC and excluded all cases with chemotherapy-induced myopathy and catecholamine toxicity as well as cases with possible infectious, ischemic, or hypersensitivity-induced myopathies. We compared the light microscopic findings of 12 TC cases of 12 cases of IDC, and measured trace Co levels on digested heart tissue samples. The TC cases had prominent myofibrillar loss and atrophy; no cases had neutrophil infiltration or frank myocyte necrosis. In contrast, IDC had minimal myofibril loss and atrophy. Cobalt levels in the range of 0.6 to 5.45 micrograms/g of dry tissue were obtained for the TC cases, while IDC demonstrated Co levels of 0.01-0.2 micrograms/g. Distinction between TC and IDC is predominantly a function of myocyte change, with TC showing myofibrillar loss and atrophy in the absence of inflammatory infiltrates and fibrosis; IDC is predominantly associated with myocyte hypertrophy, atrophy, and fibrosis.

Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry.

OBJECTIVES: The majority of HIV-1-infected individuals manifest a plethora of central nervous system (CNS) diseases unrelated to opportunistic infections, including AIDS dementia complex, encephalitis, and various other disorders of the CNS. The present study sought to evaluate the cellular reservoirs and expression patterns of HIV-1 in brain tissue to gain further understanding of HIV-1 neuropathogenesis. DESIGN: CNS tissue, obtained post-mortem from 22 patients with AIDS and four HIV-1-seronegative controls, was analyzed. METHODS: CNS samples were evaluated using a combination of in situ DNA polymerase chain reaction (PCR), reverse transcriptase (RT)-initiated in situ PCR, and immunohistochemistry. By utilizing this triple-staining methodology, HIV-1 proviral DNA and HIV-1-specific mRNA can be identified at the single cell level. RESULTS: HIV-1 was detected in all 22 AIDS brain specimens and in none of the four brains from HIV-1-seronegative individuals. The most commonly infected cells in AIDS brains were microglia cells and macrophages, but variable levels of HIV-1 infections were demonstrated in many of the major histological cell types within the CNS, including neurons, microvascular endothelial cells (MVEC) and astrocytes. The presence of HIV-1-infected cells was not uniform with infected cells unevenly distributed throughout the brain parenchyma. The degree of HIV-1 mRNA expression varied from 39-65% of the cells in the CNS harboring HIV-1 provirus. Choroid plexus and MVEC exhibited relatively high levels of productive infection. CONCLUSION: These findings demonstrate that several cell types in the CNS, in addition to microglia or macrophages, may become infected with HIV-1 in vivo with variable levels of HIV-1 mRNA expression. The diverse cellular reservoirs for HIV-1 in the CNS may be critically linked to the molecular mechanisms involved in HIV-1 neuropathogenesis. In addition, in vivo infection of MVEC, and possibly cells in the choroid plexus, may directly contribute to penetration of the blood-brain barrier by HIV-1.

Calcium oxalate crystals in human pathology. Molecular analysis with the laser Raman microprobe.

OBJECTIVE: Calcium oxalate crystals in pathologic specimens were examined by the laser Raman microprobe, a nondestructive spectroscopic technique. Although research focused on the identification of calcium oxalate deposits in tissue sections, kidney stones were also studied to determine the in situ structural specificity of the technique. DESIGN: Paraffin-embedded tissue specimens were cut into sections of 2 to 6 microns. The unstained sections were placed on metal (aluminum)-plated slides and excited with the 514.5-nm line of an argon-ion laser, which was focused to a 1-micron spot size using a high-resolution optical microscope. MAIN OUTCOME MEASURE: The laser Raman microprobe technique generates spectra that differentiate the monohydrate (CaC2O4.H2O, whewellite) and the dihydrate (CaC2O4.2H2O, weddellite) forms of calcium oxalate inclusions in tissue sections. RESULTS: Characteristic spectra were generated and provided unequivocal evidence for the identification of the dihydrate oxalate form of calcium oxalate crystals in cases of oxalosis of the myocardium and for the monohydrate oxalate structure in a case of oxalosis of the pituitary. Finally, the combined occurrence of both oxalate structures was confirmed in kidney stone specimens. CONCLUSION: The results obtained in this investigation demonstrate the efficacy of the laser Raman microprobe as a useful adjunct in diagnostic pathology.

A comparison of two commercially available in vitro chemosensitivity assays.

In vitro chemosensitivity assays (IVCAs) are expensive laboratory tests utilized to assist oncologists in the selection of chemotherapeutic regimens. Their utility is disputed; yet, these assays continue to be requested because of the importance of the information they can provide and their scientifically logical approach. Therefore, we compared the results of two assays offered to clinicians at our hospital; the extreme drug resistance assay performed by Oncotech (OT) and the fluorescent cytoprint assays performed by Analytical Biosystems (AB). The two techniques used and the expression of assay results by the two companies are discussed. Twenty neoplasms, all at least 3 cm in diameter and predominantly of breast and ovarian origin, were compared. OT performed 74 drug assays on 17 tumors, while AB performed 194 assays on the corresponding neoplasms; 3 neoplasms were insufficient for comparison. Evaluation of the results revealed apparent disagreement on at least 44 drug assays with complete disagreement on at least 2 of the drugs tested in 12 of 17 cases. In conclusion, based on available information, comparisons between IVCAs show great variation in results; prospective studies are needed to evaluate commercially available assays for correlation with clinical outcome, and results should be expressed so comparisons can be readily made. Though utility may be limited to tumors resistant to standard therapy, cost and benefit to the patient will ultimately determine the fate of these tests.

Pseudoinvasion in intraductal carcinoma.

Fine-needle aspiration, stereotactic biopsies, and guide wire localization have introduced an element of pre-excision trauma to mammary lesions. Dislodgement of tumor cells may result in diagnostic difficulties and misinterpretation of a tissue artifact as an invasive carcinoma. Eight breast biopsy specimens with intraductal carcinoma (ranging from cribriform to comedo types) displayed changes suggestive of an invasive carcinoma. Three of the patients had a prior history of needle aspiration; the remaining five women had undergone needle localization to guide the biopsy. In all cases, two or more dislodged tumor cell clusters were found in the stroma or adipose tissue either immediately adjacent to a disrupted duct with intraductal carcinoma or in the nearby stroma. Those cases with prior needle aspiration were associated with significant hemorrhage and reactive changes with small, rounded clusters of tumor cells within pools of blood. The needle localization specimens had minimal tissue reaction with larger fragments of detached cell clusters. Breast trauma by a puncturing instrument (needle or guide wire) can disrupt mammary ducts with intraductal carcinoma and dislodge the proliferating cells into the surrounding stroma. The dislodged cells simulate invasion. To minimize damage to the architectural integrity of the lesion under investigation, limits should be imposed on the number of needle passes.

Potential of the in situ polymerase chain reaction in diagnostic cytology.

Routine examination of cytologic material by light microscopy in our laboratory includes special stains and enzyme histochemistry, but molecular biology techniques are not generally employed. We examined the feasibility of utilizing the in situ polymerase chain reaction (PCR) for examination of archival cytologic specimens. Protocols were shortened in an effort to employ a technique that would be economical and have diagnostic relevance; a result would be obtained within two days of a request. Cases of transitional cell carcinoma were examined for the p53 tumor suppressor oncogene; preparation of direct incorporation PCR required eight hours of laboratory work, thermal cycling was performed overnight, and product visualization required three hours of laboratory work the following day. Amplification products were found in the cytoplasm and nuclear regions with an antidigoxigenin fluorescent and peroxidase probe. In situ PCR has enormous potential in the diagnostic cytology laboratory.

Is it safe to omit the 37 degrees C reading from pretransfusion red blood cell antibody detection testing?

The routine pretransfusion red blood cell antibody detection test (PADT), performed at the authors' institution, consists of a three-phase, saline-antiglobulin technique (immediate spin, 30-minute incubation at 37 degrees C, IgG indirect antihuman globulin test [IAT]), and each phase is examined for hemolysis and agglutination. To determine if it would be safe to omit reading the 37 degrees C phase of the PADT, a 6-year retrospective review of records (February 1986 to February 1992) was undertaken. Of approximately 280,000 sera tested for unexpected red cell alloantibodies, 1480 (.53%) were reactive at only 37 degrees C. Of 1480 sera, 1313 contained alloantibodies of no or questionable significance (eg, anti-Le(a), anti-Leb, and anti-P1), 71 sera contained antibodies of undetermined specificity, and 10 sera were reactive because of rouleaux. Eighty-six serum samples from 53 different patients contained alloantibodies of potential significance (anti-K, anti-E, etc). These 86 sera represented approximately 2.2% of all reactive sera that contained potentially significant alloantibodies. Although most antibodies (approximately 94%) detected only at 37 degrees C were of no or questionable significance, the other 6% could have increased the risk of missing a potentially significant antibody from approximately 1 in every 4700 sera tested to 1 in every 1875 sera tested, had they not been detected. The authors suggest that the increased risk of an acute or delayed hemolytic transfusion reaction would be too high to justify a procedural change. Based on these data, the authors continue to read the 37 degrees C phase of the PADT for hemolysis and agglutination.

Simple renal cysts, atypical renal cysts, and renal cell carcinoma in von Hippel-Lindau disease: a lectin and immunohistochemical study in six patients.

We compare the expression of four markers of renal tubular differentiation in six renal cell carcinomas, five atypical renal cysts, and five simple renal cysts from six patients with von Hippel-Lindau disease. Proximal tubular markers were expressed by five of six renal cell carcinomas, three of five atypical renal cysts, and zero of five simple renal cysts. Distal tubular markers were expressed by one of six renal cell carcinomas, five of five atypical renal cysts, and four of five simple renal cysts. One of the three atypical cysts which expressed distal tubular markers was associated with a renal cell carcinoma which also expressed distal tubular markers. Our findings suggest that simple renal cysts in von Hippel-Lindau disease arise more commonly from distal rather than proximal tubules, while atypical renal cysts show tubular origin similar to renal cell carcinomas.