General Comments

CLINICAL DISEASE AND POSTMORTEM CULTURE RESU LTS The medical literature is replete with examples of discrepancies between clinical evidence of infection and postmortem culture results (1-6). These discrepancies have been attributed to contamination during specimen collection (5,6), transmigration of bacteria from the gut into surrounding tissues and blood (2,4), and even the presence of indigenous bacteria in normal, healthy tissue (7). Of these explanations, contamination is most frequently implicated and remains a major obstacle in meaningful postmortem microbiology. The theory of transmigration has never been substantiated. Articles published as early as 1921 offered evidence to disprove it (8). As for the presence of indigenous bacteria in normal tissue, this theory has never gained much support.

Given the lack of specificity inherent in postmortem microbiology, it is necessary for the prosector to be very judicious in the selection of specimens for culture. This will optimize the information obtained as well as limit the cost of microbiologic assessment. Moreover, judicious use of cultures in the autopsy service will foster a good working relationship with the microbiology laboratory.

A thorough knowledge of gross pathology is the best tool for determining what specimens to submit for culture. In most patients whose immunologic status is intact, a grossly visualized host response, such as pneumonia, abscess, caseating granuloma, or collection of cloudy fluid is the best indication for culture. As a rule, one should not culture if there is no host response and no clinical information to raise suspicion of infection. Positive cultures from tissues that show no inflammation histologically generally are the result of contamination and, thus are meaningless.

CONSIDERATIONS IN IMMUNOCOMPROMISED PATIENTS These cases require a different approach at autopsy because infections may not be grossly evident. Thus, the selection of specimens for microbiological assay must be directed by a thorough understanding of the clinical history, close communication with the physicians who cared for the patient, and a carefully thought-out differential diagnosis of possible etio-

logic agents. At the autopsy table, the pathologist should carry a higher index of suspicion and thus may be prompted to submit cultures from organs that show no gross features of infection. Moreover, the variety of organisms to which these patients are susceptible is greater, requiring a larger scope of microbiological methods to isolate and characterize them. As a result, specimens might be collected for bacterial, mycobacterial, and fungal culture, and tissue submitted in transport media for viral culture. Immediate placement of tissue in fixative for electron microscopy, freezing of tissue at -70°C, and obtaining air-dried smears or touch preparations also may be indicated.

THE GRAM STAIN IN AUTOPSY STUDIES A powerful tool in the autopsy pathologist's armamentarium is the Gram stain. This stain is very inexpensive, easy to perform, and can be done by the pathologist with little inconvenience to the clinical laboratory staff. Moreover, Gram stains of touch preparations or smears demonstrate bacterial morphology much better than Gram stains of 5-pm paraffin sections. With just a brief examination of the smear, an experienced pathologist can characterize the inflammatory response or identify a neoplasia. Such findings may direct the focus of further investigation.

If the presence of an infection is in doubt, tissue or fluid samples may be submitted to the clinical microbiology laboratory with the instruction to "culture for bacteria if Gram stain shows inflammation." Although this instruction may sound vague, it encourages dialogue between the microbiology staff and the autopsy physicians; it also demonstrates the commitment of the autopsy service to minimize unnecessary cultures.

Touch preparations of tissue for Gram staining are best obtained from a 1-cm3 sample from which excess blood is removed by touching the specimen once or twice with a paper towel. Then, the tissue fragment is blotted 2 or 3 times onto different areas of a slide. Following air-drying and heat fixation, the touch preparations are ready to Gram stain and examine. The "pull-prep" method is useful in preparing smears of fluid or pus for Gram staining. In this procedure, a single drop of fluid is placed onto the center of a slide. A second slide is pressed against the first, keeping the slides essentially congruent, and then the two are pulled apart, thereby spreading the fluid into a thin layer. It is best to place the drop of fluid in the center of the slide so as to maximize the area over which the fluid will be spread. In most cases, a single small drop of fluid is sufficient.

Fig. 9-1. Equipment for processing autopsy specimens for culture. These tools should be stored and readily available in the autopsy room. See text for detailed description.

PRINCIPLES OF SUBMISSION AND CULTURE OF SPECIMENS In most situations, the use of swabs for collecting specimens is inadequate. Fluids or exudates should be collected in needle-less syringes, which may be conveniently sealed with the cap that accompanied them. Two to three milliliters of fluid are sufficient. This volume allows storage of leftover specimens for future use, should new questions arise. Some laboratories may find it useful to freeze and store leftover tissue for several weeks in case histologic examination reveals an unexpected finding, such as viral inclusions or granulomatous inflammation. Tissue specimens should measure at least 1-2 cm3. If the sample is too small, it may dry during transportation to the microbiology laboratory.

The tools needed to obtain specimens for culture at autopsy (Fig. 9-1) include a Bunsen burner, spatula, forceps that can be sterilized in the flame, scalpels, sterile syringes and needles, sterile containers and blood culture bottles, povidone iodine and alcohol swabs, an appropriate container for disposal of sharps, and the necessary writing utensils to label the containers and requisition slips. Protective eyewear (see Chapter 16) and gloves are also required. Glass slides should be available for smears and touch preparations. Any grinding or surface decontamination of tissues is best performed in a biosafety cabinet by microbiology technologists.

Finally, both safety and courtesy demand that all specimen containers departing the autopsy suite be clean and dry on the outside. Given the nature of the autopsy procedure, this standard may take more effort to achieve but it must be no less inviolable. It is often helpful to have an assistant with clean, gloved hands who can fill the blood culture bottles and handle the containers while the prosector procures the specimens.

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