Azorides R. Morales

Mutations in TNNT2 , encoding cardiac troponin T, commonly shows early onset, aggressive dilated cardiomyopathy (DCM). This observation may influence the decision of whether to undertake clinical genetic testing for TNNT2 in later onset DCM. Further, the trigger for late onset DCM remains enigmatic. A 70‐year‐old woman, previously healthy with a left ventricular ejection fraction of 50%–55% at age 69, presented with DCM of unknown cause and a 4‐month history progressive heart failure requiring cardiac transplantation. Clinical genetic testing revealed a novel TNNT2 R139H mutation but no relevant variants in 18 other DCM genes. Her explanted heart showed partial fatty replacement in the right ventricle. Sequencing for five arrhythmogenic right ventricular dysplasia genes was negative. Functional studies in porcine cardiac skinned fibers reconstituted with the mutant R139H troponin T protein showed decreased Ca 2+ sensitivity at pH 7, characteristic of DCM. Because fatty infiltration may acidify the myocellular environment, maximal force development examined at pH 6.5 was diminished, suggesting a possible environmental trigger. We conclude that the TNNT2 R139H mutation was likely to be disease causing. Further, later age of onset may not be relevant to exclude genetic testing for TNNT2 mutations. Clin Trans Sci 2010; Volume 3: 219–226.

The potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer - estrogen receptor and HER2 - at the RNA and protein levels. Parallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR. The immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens. The formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

Currently, surgeons have to wait for at least 1 day to receive the pathology report of a biopsy or other surgical excision. This delay is mandated by the overnight tissue-processing methods that have been in use for more than a century. Patient anxiety and delay in treatment are consequences of this practice. Here we report the impact of a tissue-processing system on the turnaround time of surgical pathology reporting and its potential effect on overall patient management. This technique provides the feasibility for performing molecular assays on the same sample used for pathologic diagnosis. Biopsies and other surgically removed specimens from patients treated at the University of Miami, Jackson Memorial Hospital during calendar year 2005 were processed by an automated, microwave-assisted rapid tissue-processing method. Turnaround time for surgical pathology reports was calculated and compared with that of year 1996, the last year before the new technology was phased in. Total tissue-processing time was reduced from 8 to 10 hours to 67 minutes, resulting in the availability of slides in less than 3 hours. In 80% of the patients, diagnoses were reported on the same day they were received in the laboratory. The 1-day turnaround for the reports in 1996 was < 1%. Histology of rapidly processed tissues and their histochemical and immunohistochemical properties were comparable with those of the traditionally prepared material. The rapid turnaround capability of the new tissue-processing system has allowed the pathology laboratory to render the final report in the majority of specimens on the day they are received. The feasibility of preserving macromolecules in the same clinical samples used for diagnosis is a timely advantage in the era of molecular medicine.

To determine whether universal molecular fixative (UMFIX), a novel human tissue fixative, could also be used as an animal tissue fixative for histomorphology and a preservative of RNA at subtropical temperatures. Cat, dog, mouse, pigeon, rabbit and rat tissue, as well as ant, beetle, earthworm and lizard were collected. Tissue was fixed in UMFIX for up to a week at room or ambient temperatures, processed and paraffin embedded. Histomorphology and RNA quality were evaluated and compared to formalin-fixed and fresh frozen tissue. Animal tissue fixed in UMFIX at room temperature or high ambient temperature (30–34 °C) provides similar histomorphology. Comparable to other alcohol-based fixatives, UMFIX produces a histomorphology similar but not identical to formalin. All minor histopathological differences, however, in no way interfere with establishing the correct diagnostic conclusion. Whereas RNA extracted from animal tissue fixed in formalin was completely degraded, tissue fixed up to 1 week in UMFIX at high ambient temperatures rendered completely intact RNA. UMFIX represents a new class of preservative/fixative that protects RNA and provides acceptable histomorphology. Tissue fixation and preservation of RNA can be achieved at high ambient temperatures. This allows collection of animal tissue in field research without the need for immediate freezing of tissue and also provides histomorphology comparable to formalin-fixed tissue. Furthermore, as RNA is also preserved in UMFIX preserved paraffin-embedded tissue, if amount of tissue is limited the same tissue that is used to determine histomorphology can be used to extract RNA.

Laser-capture microdissection techniques have enhanced the ability to perform molecular studies of pure-cell populations. Although many technical factors affect the outcome of the procedure, none is more critical than the appropriate handling of the tissue. Because extraction of intact RNA from paraffin-embedded tissue is a difficult and inconsistent process, frozen sections with their attendant problems are used for this purpose. The major limitation of frozen section is its inferior morphologic quality compared with paraffin-embedded sections that may complicate accurate identification of cells during microdissection. We have developed a procedure that provides both high-quality histomorphology and RNA preservation in paraffin-embedded tissue. It is based on the use of a methanol-based fixative coupled with microwave-assisted rapid tissue processing. This technology in conjunction with a modified hematoxylin-eosin stain and a RNA extraction method allows isolation of high molecular-weight RNA from laser-capture microdissected, hematoxylin and eosin-stained paraffin sections. The high quality of the extracted RNA was confirmed by capillary electrophoresis and RT-PCR. The combination of a methanol-based fixative, rapid microwave tissue processing, and a modified hematoxylin and eosin stain produces paraffin sections that yield high molecular-weight RNA upon microdissection. This methodology opens the door for a wide range of gene expression analyses using paraffin-embedded tissue.