Supplemental Laboratory Studies

Collection of Samples

The hospital autopsy pathologist should take full advantage of modern chemical, microbiologic, cytogenetic, and molecular analysis of body fluids, tissues, and cells as a supplement to anatomic dissection and microscopic examination if necessary. Careful review of the case records usually indicates to the prosector whether special studies are needed. In most instances, the collection and proper storage of samples for such studies requires little in the way of additional work for the pathologist and offers the possibility of valuable diagnostic information. However, proper acquisition of diagnostic material and appropriate analytic methods are keys to obtaining reliable results. In addition to collecting samples at autopsy, pathologists should also consider asking the clinical laboratories to hold any available antemortem samples for subsequent analysis.

Postmortem Microbiology

Microbiologic analysis has become increasingly important in hospital-based autopsies, and this is augmented by recent improvements in laboratory techniques. Factors affecting the accuracy of microbiologic studies performed on postmortem fluids and tissues include the postmortem interval (though less than might be expected), promptness of cooling, degree of organ manipulation before sampling, technique of obtaining the sample, and therapy with antibiotics prior to death. Interpretation of the results obtained from a positive microbiologic culture must be correlated with the patient's history and gross and microscopic pathology findings. Nichols emphasized the utility of preparing smears for Gram staining to aid in the interpretation.

Obviously, the best postmortem culture results are obtained from grossly infected tissues and body cavity fluids rather than from routine cultures of fluids or tissues. If possible, tissue specimens should be obtained in situ with the least manipulation of other organs. For example, when obtaining lung cultures, it may be done immediately after removal of the chest plate and before the peritoneal cavity is opened. To culture tissue from an organ, one should first clean and dry the surface, and traditionally the surface is sterilized by searing the organ surface with a hot spatula. Alternatively, to prevent potential aerosols containing infectious organisms, the surface can be sterilized with an iodine-containing disinfectant and a deep piece of tissue obtained with a sterile scalpel and forceps, taking care not to include any tissue exposed to disinfectant. The tissue (1 cm ³ ) is placed in a sterile plastic cup and submitted for viral, bacterial, or fungal culture. Abscesses and granulomata should be cultured from both their center and periphery and instruments should be changed or flame sterilized before sampling different sites. Fluid in a body cavity can be collected using a sterile syringe and needle. Purulent material in a body cavity or from an abscess can be swabbed with a sterile, cotton-tipped applicator. If meningitis is suspected, CSF should be obtained as described previously. Swabs should also be taken from the subarachnoid space. The subarachnoid space is reached by lifting the leptomeninges and making a small cut into the meninges.

Blood cultures often yield mixed growth of bacteria that are nondiagnostic. However, if the body is refrigerated at 4°C to 10°C shortly after death, postmortem growth of endogenous microorganisms is delayed. In fact, postmortem interval has only a minimal effect on the isolation rate of blood cultures. Postmortem blood cultures are generally obtained from the inferior vena cava after the pericardium is opened and the heart is reflected upward. Using aseptic technique, the pathologist introduces a needle into the right atrium and aspirates blood into a sterile syringe. However, Hove and Pencil demonstrated that during the early postmortem period, percutaneous, aseptic sampling of subclavian blood provides more reliable samples. Roberts found good correlation of postmortem cultures of spleen with clinically documented bacteremia.

In many laboratories, microbial identification has shifted away from standard culture to molecular tests. At our institutions, tissue samples are tested for viral pathogens by panels of polymerase chain reaction (PCR)–based molecular tests for most common pathogens, while culture is reserved for subgroups of rare pathogens such as enteroviruses. Studies of PCR-based testing for respiratory viruses in lung tissue from autopsies of children and adults have indicated that the PCR-based testing is more sensitive than viral culture and appears to have excellent specificity as verified by clinical scenario and lung histopathology. While most bacterial and fungal identification is by standard culture, identification is also possible by 16S ribosomal RNA gene sequencing from either fresh tissues or paraffin-embedded tissues. A common workflow at our institutions is to perform 16S sequencing on appropriate portions of paraffin blocks only when cultures are not informative and histologic sections show presence of bacteria or fungus together with a clinically consistent inflammatory response.

Despite the difficulties associated with postmortem microbiology studies, their use should not be discouraged. The autopsy pathologist who takes the time to submit appropriate specimens to the microbiology laboratory is increasingly rewarded with information that not only improves the final autopsy report, but also provides vital information to hospital infection control personnel and local public health authorities.

Postmortem Cytogenetic and Molecular Studies

In cases of dysmorphic fetuses/infants, fetal demise, or developmental delay, careful anatomic dissections may be aided by analysis of chromosomes or genomic DNA. This is a rapidly evolving area in which communication with the clinical medical genetics consultant will help guide which specimens to submit and which tests to order ( Table 10-1 ). In general, such tests can be either broad surveys for genomic/chromosomal defects or focal tests for specific abnormalities as guided by the clinical findings and autopsy morphologic findings. For routine karyotyping, a fresh, sterile, fibroblast-rich tissue sample (skin, diaphragm, internal fascia) should be placed in RPMI or other appropriate tissue culture medium for transport to a cytogenetics laboratory. Short-interval storage should be in a refrigerator at 4°C. Because initiating successful fibroblast cultures is time sensitive, the pathologist should work with his or her colleagues in obstetrics to ensure prompt collection of appropriate samples.

Advances in molecular biology research have quickly transformed medicine and provided many useful diagnostic tests that are becoming essential components of pathologic analysis. Molecular techniques such as PCR, array hybridization, and high-throughput sequencing allow identification of germline mutations, somatic mutations (particularly in neoplasia), infectious microorganisms, and contaminating DNA from forensic evidence collected from the body of the deceased. Newer techniques often allow analysis of archived paraffin tissue blocks, but fresh or frozen tissue is usually preferred. With adequate preservation and handling, intact mRNA can be isolated from autopsy tissues and accurately profiled. Kapur strongly advocated routinely freezing a sample of liver and placenta from every fetal and perinatal autopsy case and selected tissues as dictated by the gross autopsy findings. Samples should be collected carefully and as soon after death as possible. Tissue samples for nucleic acid analysis can be snap frozen and stored at −70°C. Recently, there has been growing interest in molecular diagnosis in the setting of postmortem analysis of sudden cardiac death, as discussed in Chapter 12 . In those cases, molecular testing is not a substitute for a thorough autopsy, but it is a helpful adjunct when there is suspicion of a familial cardiomyopathy, arrhythmogenic disorder, or hyperlipidemia.

Genetic/Metabolic Disease Autopsy

Genetic metabolic disorders or inborn errors of metabolism are a large (greater than 400) group of diverse diseases that may cause death in the fetal or perinatal period ( Box 10-1 ). In other instances, genetic metabolic disorders may lead to sudden unexpected death in infants and children and become the jurisdiction of the medical examiner and coroner systems. In either case, autopsy diagnosis becomes critical not only for elucidating the cause of death, but also for providing critical information needed in subsequent genetic counseling of the parents. Ideally, the genetic metabolic autopsy (or at least the specialized fluid and tissue collections) should be performed within 2 hours of death. In the hospital setting, this requires cooperation among the clinical, pathology, and decedent affair services and readiness to perform the initial specimen collection and processing, but it is important to verify that a valid autopsy consent has been obtained before any postmortem intervention or dissection. Table 10-2 lists the major components that may be part of a genetic metabolic autopsy. In practice, only a subset of the listed fluid and tissue sampling is performed in most cases, as guided by the clinical team and pediatric medical geneticist.