Routine Sampling Of Toxicologic Material

In the author's office, it is usual practice in all autopsies to save 50 mL each of central blood, bile, urine, liver, and brain, plus available femoral vein blood up to 50 mL, and all retrievable vitreous. Approximately half the blood is placed in containers with sodium fluoride as a preservative. Sodium fluoride inhibits both bacterial growth and serum esterases which hydrolyze cocaine postmortem. If commercially available gray-top Vacutainer® tubes are not used, 250 mg of NaF can be added to 30 mL containers.

Urine Urine is aspirated with a syringe through the dome of the bladder after the peritoneal cavity has been opened. If the bladder is nearly empty, it can be secured by hemostats before incising the dome to facilitate aspiration of the bladder lumen under direct vision. Toxicologists often prefer urine as a specimen for enzyme-multiplied immunotechnique (EMIT) and enzyme-linked immunosorbent assay (ELISA) drug screening, because it can be analyzed without extraction. NaF as a preservative is optional; as an inhibitor of cocaine hydrolysis NaF is unnecessary, because the immunoassays detect cocaine metabolites rather than parent cocaine.

Blood Central luminal blood is preferred to cavity (pleural, pericardial, or peritoneal) blood. Central ("heart blood") specimens are aspirated from any chamber of the heart, or from the intrapericardial thoracic aorta, pulmonary artery, or vena cava. However, for a growing number of analytes, most notably tricyclic antidepressants, peripheral blood is preferred over central blood. Peripheral blood is aspirated by percutaneous puncture before autopsy, from the femoral vein or the subcla-vian vein. The author prefers the femoral approach in order to avoid any question of artifact in the diagnosis of venous air embolism. Peripheral blood can be obtained by a technician as soon as the body is received. If cocaine intoxication is likely, it is highly desirable to obtain this specimen in a tube with NaF as soon as possible, in order to inhibit postmortem hydrolysis of cocaine. The term, "cavity blood" is used for blood ladled or aspirated from a hemothorax, hemopericardium, hemoperito-neum, or from the pooled blood left in the common cavity after removal of the heart and lungs. Cavity blood analyses should be supplemented by peripheral blood, vitreous, or solid tissue analyses, because of the real possibility of contamination from gastric contents.

Vitreous Vitreous is an excellent specimen for alcohol and drug analysis. The protected location in the orbit renders the fluid less susceptible to putrefaction than blood, and the problem of site-dependent variation in concentrations in blood specimens is avoided. Two to three mL of vitreous from one or both eyes is gently aspirated from the lateral angle of the eye with a 5 mL clean syringe. The tip of the needle should lie near the center of the eyeball. The procedure is illustrated in Chapter 7 (Fig. 7-1). Forceful aspiration must be avoided because it may detach retinal cells, which cloud the specimen and give spuriously high potassium values. Before dilution, the chemist must invert the specimen 10 or 12 times to ensure thorough mixing.

Gastrointestinal Tract After removal of the stomach, duodenum, pancreas, and esophagus, the gastric contents are squeezed out through the esophagus, or through an incision in the stomach, into a 1-L container. A representative 50-mL specimen is satisfactory for the toxicologist, unless the stomach contents have a nonuniform slurry of solid and liquid elements, in which case a higher volume is desirable. If the solid elements seem to be fragments of medicaments, then nearly all the contents should be saved for the toxicologist, except for what is needed to strain and inspect the material to identify food matter. In suspected suicides, in which death may have followed ingestion by several hours, it can be useful to ligate a length of jejunum before removing it and draining it into a specimen container. The jejunum in such circumstances may have a higher concentration of analyte than the stomach.

The establishment of toxicity in adults cannot be done from analysis of gastric content; investigative information and analysis of tissue or body fluids are needed. Analysis of gastric content may help to establish suicidal intent and to investigate poisoning in infants. In infants, screening of gastric contents also can be used to save the limited quantities of blood for quantitative analysis.

Cerebrospinal Fluid (CSF) The practice of removing CSF (see Chapter 6) by suboccipital or lumbar puncture is mentioned only to discourage it. Although pathologists certainly vary in their skill levels, and some can make a clean puncture more often than not, even in the best hands blind punctures often produce blood-lined tracks that render the interpretation of posterior neck and vertebral dissections problematic. Vitreous, like CSF, is a low-protein erythrocyte-free substitute for blood, and is preferred in most situations. If CSF must be drawn, it is best taken from the cerebral cisterns after the skull has been opened is such a fashion that the leptomeninges are relatively intact and the CSF has not run out. The situation most often calling for a CSF specimen is the meningitis autopsy with no urine available for a latex agglutination test for bacterial antigens.

Bile Bile is aspirated by needle after the abdomen is opened and before the organs are removed. Because the mucosa of the gallbladder is lush and easily becomes ensnared in the needle tip, it is helpful to aspirate with gentle vacuum, and to use the free hand to milk the gallbladder. Bile is a useful substitute for blood when the analyte of interest is an opiate or an alcohol. In rapidly fatal opiate intoxications, the offending opiate may be detectable only in bile.

Other Liquid Specimens In hospitalized decedents, the highest concentrations of toxic substances may be found in dialysis and lavage fluids, if they have not been discarded after death.

Solid Organs Liver is the solid organ of choice when no liquid specimens are available. Reference values are available for the lethal concentrations of numerous types of drugs in liver tissue. Liver specimens from the right lobe of the liver are preferred to specimens from the left lobe, to avoid spuriously high concentrations from diffusion from the stomach (22). Brain tissue is useful for alcohol determinations in the absence of a useful liquid specimen.

In putrefied bodies, blood and bile are usually absent, and the only specimens available may be solid organs, such liver, brain, and skeletal muscle. Skeletal muscle from the least decomposed extremity is preferred.

In fire deaths, arson investigators occasionally request specimens for accelerant analysis. For this purpose, lung tissue is sealed in an unused lidded metal can of the style used by paint manufacturers.

Hair Hair is a useful specimen in suspected chronic arsenic poisoning, and may be useful in the determination of chronic drug abuse. Hair should be pulled from the scalp, to include the roots. A large sample, about 10 grams, should be tied in a lock to identify the root end of the specimen.

Skin If it is suspected that a poisonous substance has been injected, the skin around the needle-puncture site can be excised at a radius of 2-4 cm from the injection site. If a poisonous substance might have been taken up by absorption, the skin is excised in the area where the absorption is thought to have occurred, and from a distant, preferably contralateral area as a control. Skin samples are saved with the expectation that the toxicologist will prefer to obtain the information necessary to opine the cause of death by first analyzing the customary liquid specimens.

CHAIN OF CUSTODY The continuity of the custody of the specimens should be documented. A blank space on the specimen transmittal form (see next paragraph) can be used for tracking custody from the pathologist to an in-house toxicology laboratory.

Transmittal Sheet Specimens submitted to a toxicology laboratory should be accompanied by a transmittal sheet and a summary of the investigative information as it is known at that time. The transmittal sheet contains case identifying information, a list of specimens, supplementary information necessary to select analytical methods, and signatures to indicate the chain of custody. It should state whether the body is embalmed or decomposed and indicate the duration of hospitalization (during which an alcohol or drug is metabolized). An example is in Table 2-3.

If a courier is used to transport a sealed container with multiple specimens from multiple cases to an outside laboratory, an additional, separate, single transmittal form can be devised that lists all the case numbers; omits specimen details; and has signature/date lines for the in-house technician, the courier, and the receiving clerk at the laboratory.

METHODOLOGY Although the techniques of toxico-logic analysis are beyond the scope of this book, a brief summary of current methods is in order.

Volatiles by Gas Chromatography The analyte most frequently tested is ethyl alcohol. Toxicologists in medical examiner offices generally detect and quantify ethyl alcohol by gas chromatography, as part of a general panel designed to capture numerous volatile compounds, including ethyl, methyl, and isopropyl alcohols, and ketones. Tertiary butyl alcohol is often used as an internal standard, because it does not occur naturally. Hospital and clinical laboratories most often use the alcohol dehydrogenase method, which measures any substance capable of being dehydrogenated by the enzyme. It does not distinguish methyl, ethyl, and isopropyl alcohols and it has a larger experimental error than does gas chromatography. The dichromate method, which measures oxidizing activity, is nonspecific and mainly of historical interest.

Specific Drug Screening by Enzyme-Multiplied Immunoassay (EMIT) Drugs of abuse are commonly detected but not quantified by EMIT (Enzyme Multiplied ImmunoTechnique), in which the activities of selected families of drugs are measured by antibody interaction. The panels are selected depending on local drug-abuse patterns. Panels are available for cocaine metabolites, tricyclic antidepressants, barbiturates, cannabin-oids, amphetamines, opiates, and propoxyphene. Not detectable are drugs present in parts per billion, such as fentanyl.

Specific Drug Screening by Enzyme-Linked Immunosorbent Assay (ELISA) Gradually supplanting the EMIT technique is the ELISA technology, which also uses antibodies, but is capable of detecting drugs whose concentrations are in parts per billion.

Drug Screening by Thin-Layer Chromatography (TLC)

Although EMIT and ELISA panels detect the most commonly occurring abused drugs, they are not general drug screens. The technically simplest general drug screen utilizes the TLC so familiar to high school chemistry students. Specimens are prepared for TLC by extracting into solvents under acidic, neutral, or basic conditions, in order to bring different classes of drugs into the extraction solvents.

General Drug Screening, Identification and Quantitation by High-Performance Liquid Chromatography (HPLC) Supplanting TLC is high-performance liquid chromatography, in which the chromatograph is a thin column with packing material and a liquid solvent. HPLC can be linked to a computer database of hundreds of drugs to provide spectral identification and quantification. Historically, HPLC has been used by most laboratories for assaying specific classes of drugs such as tricyclic antidepressants. A few laboratories have developed extraction methods and columns that permit HPLC to be used as a general screen. HPLC, with its cool injection ports, is often a preferred quantitative method when compared to gas chro-matography/mass spectrometry (GC/MS) (see below), which uses hot injection ports in the gas chromatograph to volatilize drugs. The heat decomposes drugs such as methocarbamol and propoxyphene.

Specific Drug Identification and Quantitation by Gas Chromatography (GC) Linked to Mass Spectrometry (MS) The gold standard for identifying drugs is gas chromatography linked to mass spectroscopy (GC/MS). GC utilizes a gaseous medium to separate the analyte drugs in a column. The output of the column is fed into a mass spectrometer, which breaks compounds into ionic subunits, whose weights form a bar-graph spectrum that can be specific for each compound.

Carbon Monoxide Tests Carboxyhemoglobin is detected in most medical examiner toxicology laboratories by visible spec-trophotometry. In hospitals, carboxyhemoglobin is frequently detected and reported in the course of routine arterial blood gas analysis. Some medical examiner laboratories use GC for the determination of carboxyhemoglobin.

Metals Heavy metals can be detected by qualitative tests. For example, the Reinsch test primarily detects arsenic, and is an insensitive test for mercury, antimony, and bismuth. Quantifica-

Table 2-3

Toxicology Specimen Transmittal Sheet

Toxicology Specimen Transmittal Sheet (Name of Medical Examiner Agency)

(Address and Telephone Number of Medical Examiner Agency)

Medical Examiner Case Number: 97-012345 Name of Decedent: Joe Doe Date Specimens Obtained: 5/3/97

Duration of Hospitalization: 36 hours Embalmed? No Decomposition (circle): None 1+ 2+ 3+ 4+ Check here to retain specimens and issue a report that states "Toxicology Testing Not Indicated" X

X Blood, hearf X Blood, peripheral Blood, cavity

Liquid from heart or vessels (embalmed) X Bile X Urine X Gastric content Bowel content X Vitreous X Liver Lung X Brain Kidney

Skeletal muscle Other:_

Other information or instructions:

Pathologist name and date:

Laboratory receipt of specimen; name and date:

aAn "X" indicates specimen collected.

tion and specific metal identification is done by atomic absorption spectroscopy, usually by a reference laboratory.

Cyanide A good screen for cyanide is the nose of a person who is capable of smelling the ion. Because only a minority of persons can smell cyanide, it is helpful to know in advance if any person in an office or laboratory can smell cyanide. Textbooks state that hydrogen cyanide gas smells like bitter almonds; forensic pathologists who can smell the compound state that it has its own specific odor, which is not comparable to any other (Davis JH, personal communication, 1984).

SAMPLING FOR SPECIFIC TOXICOLOGIC SUBSTANCES Pertinent procedures have been listed in Part II, under the name of the substances involved, from "Alcohol Intoxication and Alcoholism" to "Poisoning, Thallium."

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