PubMed · 7532269
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G F Babcock, S M Dawes. 1994. Immunophenotyping using fixed cells.. https://doi.org/10.1016/s0091-679x(08)61710-0
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STUDY DESIGN: Twenty-five formaldehyde solution-fixed, paraffin-embedded tissue blocks from vertebral biopsy specimen materials with presumptive diagnosis of tuberculous spondylitis and nonspecific vertebral osteomyelitis were studied. OBJECTIVES: To evaluate the sensitivity and specificity of polymerase chain reaction in detecting Mycobacterium tuberculosis in formaldehyde solution-fixed, paraffin-embedded tissue samples from histologically proved tuberculous spondylitis. SUMMARY OF BACKGROUND DATA: Diagnosis of a mycobacterial infection is a long and tedious process; because of the slow growth rate of mycobacteria on solid media, identification and antibiotic sensitivity testing can take up to 10 weeks, but the sensitivity of culture can be as low as 50%. Direct microscopy is insensitive because clinical samples may contain only few organisms. Recently, polymerase chain reaction has been applied in the rapid amplification and identification of many organisms, including mycobacteria. METHODS: The DNAs were extracted from 25 paraffin-embedded tissue blocks. An insertion element IS 6110 (Integrated DNA Tec. Inc., Corrallville, IA), a DNA sequence unique to Mycobacterium complex (M. tuberculosis and the subspecies Mycobacterium bovis), was amplified by polymerase chain reaction. Polymerase chain reaction results were compared with those of Mycobacterium culture, acid-fast bacilli staining, and histologic findings. RESULTS: Polymerase chain reaction was positive in 18 cases of 19 tuberculous spondylitis. Three of the polymerase chain reaction test results were positive with concomitant negative culture and positive acid-fast bacilli staining. There were six chronic nonspecific infections, and polymerase chain reaction results were negative in five cases; in the single positive case, DNA amplification results remained positive even after three repeated tests. CONCLUSION: Polymerase chain reaction has a sensitivity of 94.7%, specificity of 83.3%, positive predictive value of 94.7%, and a negative predictive value of 83.3%. Accuracy was calculated as 92%.
In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.
Ruthenium tetroxide and osmium tetroxide were compared as post-fixatives in the preparation of human epidermis for transmission electron microscopic examination. Both reagents revealed characteristic lamellar granules within the granular layer and extruded lamellar granule contents in the upper granular layer. The transformation of the granule contents into multilamellar sheets at the interface between the granular and cornified layers and the persistence of these sheets through all levels of the stratum corneum were demonstrated only with ruthenium tetroxide fixation. Therefore, the reactivity of osmium tetroxide with isolated epidermal lipids was examined. The failure of osmium tetroxide to reveal membrane structures in the stratum corneum can be explained by its inability to react with many of the lipid components of these membranes, rather than to selective removal of lipids during tissue processing, as was formerly believed. Ruthenium tetroxide, a stronger oxidizing agent than osmium tetroxide, overcomes this problem but has other severe limitations as a post-fixative.