Antimicrobial activity of UMFix tissue fixative.

AIMS: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms. The UMFix solution was compared with 10% neutral buffered formalin. METHODS: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin. RESULTS: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses. Bacterial spores were resistant to killing by UMFix. All organisms were killed by the 10% neutral buffered formalin preparation. CONCLUSIONS: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species. The methanol content of the fixative was responsible for the killing effect of this fixative. No killing was seen when polyethylene glycol was used alone.

Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system.

A recently introduced histologic fixative (Universal Molecular Fixative [UMFIX]) has been shown to preserve macromolecules in tissue at ambient temperature. When UMFIX-exposed tissues are processed by a formalin-free, microwave-assisted rapid processing system, the resulting paraffin blocks retain good histomorphology and intact nucleic acids suitable for expression microarray analysis. Because UMFIX may be used as an alternative to formalin, the authors set out to study the effect of this new fixation and processing system on immunohistochemistry (IHC) by analyzing a range of human neoplastic and non-neoplastic specimens. Parallel slices from surgically removed specimens were fixed in formalin and UMFIX and processed in a rapid microwave-assisted tissue processor. IHC was performed following routine procedures. The staining for those antibodies that normally required antigen retrieval was carried out with and without that step. The intensity and pattern of reactions were compared in 144 tissue samples fixed by the two methods using 70 monoclonal and polyclonal antibodies. The intensity of IHC reactions for most cytoplasmic antigens was generally equal or stronger in UMFIX tissues. This was particularly true with intermediate filaments and HercepTest, where the antigen retrieval step became unnecessary. Conversely, there was a decrease in the intensity of reactions for HepPar1, bcl-2, and three nuclear antigens (Ki-67, TTF-1, and estrogen receptor). Increasing their exposure times optimized the sensitivity of the latter four antibodies. The study shows that IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens. There is an enhancement of the sensitivity of few antibodies in UMFIX-exposed tissue, rendering antigen retrieval unnecessary. This increased sensitivity may be due to the effect of eliminating formalin from fixation and processing or the microwave energy.

Clear cell variant of thyroid carcinoma.

Three carcinomas composed of a variable proportion of clear cells, oxyphil cells, and cells with combined oxyphil and clear cell features are reported. Cytologically, these tumors were included in the category of oxyphil cell variant of follicular carcinoma. In regard to pattern, two were entirely follicular, and one had follicular and papillary areas. The biologic behavior of these tumors, however, was consistent with that of follicular carcinomas. The clear cell change could be the result of chronic TSH overstimulation. This may explain the variable histological patterns, nuclear features, and biologic behavior associated with these tumors. These tumors bear a striking histologic resemblance to metastatic renal cell carcinoma and clear cell neoplasms from other sites. Immunocytochemical stain for thyroglobulin proved to be a specific and sensitive method for identification of these tumors.

Frequency and pattern of p53 gene mutation in a cohort of Spanish women with node-negative breast cancer.

Ethnic, racial and regional differences in the frequency and pattern of p53 gene mutations have been well documented. Some of these differences have been shown to have an impact on the survival of patients with breast cancer. In this study we explored the frequency and pattern of p53 abnormality in a cohort of Spanish women with node-negative breast cancer using PCR, subcloning and DNA sequencing of archival tumors. One hundred and seventy-eight cases of breast cancer diagnosed between 1981 and 1986 at the University of Oviedo Hospital in Oviedo, Spain were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on subcloned PCR-amplified DNA, extracted from formalin-fixed, paraffin-embedded tumors. Appropriate positive, negative, PCR, and polymerase controls were utilized and evaluated. Duplicate samples of the genomic DNA were re-evaluated on all cases showing more than one mutation. One hundred and five out of 178 breast cases (59%) carried one or more p53 gene mutations. Mutations were distributed randomly from codon 128 to 305. There were 123 (88%) transition, 10 (7%) transversion, 5 (3.5%) splice junction mutations, and 2 (1.5%) deletions. Eighty-three cases (61.5%) had missense mutation, 45 (33.5%) silent, 5 (3.5%) nonsense and 2 (1.5%) frameshifts. Eighty (75%) of 120 transitions were G:C to A:T, 11 (25%) of which occurred at CpG sites. Sixteen mutations were in novel codons not reported in breast cancers previously. Codons with the highest frequency of mutations in this group were 278, 273, 213 and 227. We also detected 27 tumors with more than one mutation within a single exon or in different exons in the same patient. These findings suggest that the frequency and pattern of p53 mutations in this group of Spanish women with breast cancer is different than those reported in the United States and Northern Europe.

Thyroglobulin in carcinoma of the thyroid: an immunohistochemical study.

To determine the value of thyroglobulin as an immunohistochemical marker for thyroid neoplasms, we studied 42 primary thyroid carcinomas, 38 metastatic carcinomas, and four sarcomas involving the thyroid gland. All follicular and papillary carcinomas, regardless of their morphologic variation, stained positively for thyroglobulin, whereas the medullary carcinomas, metastatic tumors, and sarcomas showed negative staining reactions. The only small-cell variant of follicular carcinoma and ten of 14 spindle and giant cell carcinomas showed the lowest thyroglobulin reactivity. It is concluded that immunohistochemical demonstration of thyroglobulin is a sensitive and specific method of identifying thyroid carcinomas of follicular cell origin.

C-cell hyperplasia in thyroid tissue adjacent to follicular cell tumors.

An immunohistochemical study was conducted on the number and distribution of C-cells in the nonneoplastic thyroid tissue adjacent to tumors of follicular cell origin. It consisted of 49 cases, of which 25 were papillary carcinomas, 22 were follicular adenomas, and 2 were follicular carcinomas. Twenty normal adult thyroids from the Broward's Medical Examiner's morgue served as controls. In 17 of the 49 cases (34.6%), there was a statistically significant increase in the number of C-cells in the normal-appearing thyroid tissue adjacent to follicular cell tumors, with at least 50 C-cells in one low power field, while only one of 20 normal thyroids had a similar number of cells. (P = .02; chi 2 = 5.05). In two tumor cases there were more than 100 C-cells in several low power fields with formation of small C-cell nodules similar to those described in the type II Multiple Endocrine Neoplasia Syndrome (MEN). It was concluded that the nonneoplastic thyroid tissue adjacent to 34.6% of tumors with follicular cell phenotypes contains significantly more C-cells than those present in normal adult thyroids. The possible pathogenesis and clinical significance of these findings are discussed.

Cathepsin D in host stromal cells, but not in tumor cells, is associated with aggressive behavior in node-negative breast cancer.

One hundred fifty-four axillary lymph node-negative invasive ductal carcinomas of the breast were immunohistochemically evaluated for the expression of cathepsin D. Formalin-fixed paraffin sections of each tumor were stained using a polyclonal antibody raised against recombinant procathepsin D. Cathepsin D content of tumor cells and host histiocytes and fibroblasts within or immediately at the invasive border of tumors were assessed separately and correlated with histomorphology, estrogen-receptor content, and patients' survival data. Positive cathepsin D staining of tumor cells was associated with a lower nuclear grade and well-differentiated histology, whereas moderate to strong staining of host cells correlated with larger tumor size, higher nuclear grade, poorly differentiated histomorphology, and lack of estrogen-receptor (ER) protein. No statistically significant correlation was found between cathepsin D in tumor cells and survival. There was, however, a statistically significant correlation between moderate to strong cathepsin D staining of host cells and shorter disease-free and overall survivals. Expression of cathepsin D by host cells, however, did not have an independent influence on survival. The authors conclude that cathepsin D in stromal cells, but not in tumor cells, is associated with aggressive behavior in node-negative invasive ductal carcinomas of breast. Furthermore, determination of cathepsin D in cytosolic extracts of tumors is of no practical value because it may represent cathepsin D content of tumor cells, intratumoral host cells, or both.

Intramural left anterior descending coronary artery: significance of the depth of the muscular tunnel.

To establish whether an intramural left anterior descending coronary artery (LADA) is a simple anatomic or a singularly pathologic variant we studied 39 hearts, each with an intramural course of the LADA and no coronary artery disease, valvular derangement, cardiomyopathy, or congenital anomaly. Seventeen of the 39 hearts had no myocardial lesions, while 22 had gross and/or microscopic alterations in the myocardial territory supplied by the intramural LADA. The myocardial lesions consisted of one or more of the following: interstitial fibrosis, replacement fibrosis, contraction band necrosis, and/or increased vascular density in areas of focal fibrosis. The coronary anatomy of the 22 hearts with myocardial lesions (group 1) was compared with that of the 17 hearts without myocardial changes (group 2). Each of the group 1 hearts had an intramural LADA deeply placed within the ventricular wall and attenuation of potential collateral blood flow because of a co-existing intramural course of the posterior descending artery, other epicardial coronary arteries, and/or a diminutive right coronary artery. The myocardial changes in group 1 hearts and their absence in group 2 hearts suggest that the deep, intramural LADA of the group 1 hearts is abnormal rather than a simple anatomic variant of normal. Furthermore, the deep intramural LADA may be associated with sudden death since 13 of the 22 group 1 hearts were from sudden death victims. Six of these 13 persons died suddenly during vigorous exercise.

Hemangioblastomas: histogenesis of the stromal cell studied by immunocytochemistry.

Twenty-one cases of hemangioblastoma from the cerebellum, spinal cord and retina were studied using the unlabeled antibody peroxidase-antiperoxidase technique with antibodies directed against glial fibrillary acidic protein (GFAP) and factor VIII related antigen (VIIIR:Ag). In 19 of 21 cases studied with anti-GFAP, astrocytes were identified peripherally, and in 13 cases they were found centrally within the tumor. In no instance did stromal cells react positively for GFAP. Sixteen cases with anti-VIIIR:Ag antibody were examined, and in all cases many stromal cells showed positive staining. It is concluded that the stromal cells were of endothelial origin. The occasional stromal cells that other investigators have identified as reacting positively for GFAP may represent stromal cells capable of ingesting extracellular GFAP derived from reactive astrocytes within the tumor, or they may be lipidized astrocytes.

Nocardia asteroides aortitis with perforation of the aorta.

A 53-year-old man died of nocardial aortitis eight months after undergoing aortic valve replacement. Autopsy revealed vegetation in the area of a previous aortotomy incision and a small perforation of the aorta enclosed by pericardium immediately above the prosthetic valve ring. Prosthetic valve endocarditis was not present. Multiple splenic infarcts, microabscesses of both kidneys, and myocarditis were identified. Antemortem blood cultures and postmortem cultures of the aortic vegetation grew Nocardia asteroides.

Myxoid heart disease: an assessment of extravalvular cardiac pathology in severe mitral valve prolapse.

Because of the microscopic features of the affected leaflets in mitral valve prolapse (MVP), myxoid degeneration of the valve is a common pathologic designation applied to this condition. We undertook this study as a means of gaining an insight into the occurrence and prevalence of extravalvular cardiac alterations in hearts with severe MVP. Tissues of 24 hearts with severe myxomatous transformation of the mitral valve as the sole cardiac abnormality were examined. Eighteen of the 24 subjects with severe MVP died suddenly. Only two of these had pathologic evidence of severe mitral insufficiency. Twenty-four normal hearts served as controls. The two groups of hearts came from victims of homicide, suicide, accident, or natural death. Sections of the mitral valve, working myocardium, conduction system, and cardiac nerves and ganglia were studied by routine and special connective tissue and proteoglycan stains. Similar to the findings in severely affected mitral valves, prominent deposits of proteoglycans in neural and conduction tissue readily distinguished hearts with myxomatous valve changes from the control hearts. We conclude that the commonly recognized local derangement of valvular tissue in MVP is but one specific reflection of a more general myxomatous alteration in cardiac connective tissue.

Myocardial alterations associated with aortocoronary saphenous vein bypass.

Clinical observations and gross and microscopic morphology of the hearts of 50 patients who dies at various intervals following aortocoronary saphenous vein bypass procedures are presented. Time of total cardiopulmonary bypass varied inversely with survival time among these patients. Myocardial necrosis with contraction bands and/or hemorrhagic myocardial necrosis was observed in the hearts of 44 of the 50 patients. Pathogenesis of the acute myocardial necrosis with contraction bands was multifactorial: anoxic arrest, cardiotonic drugs, electrical defibrillation, electrolyte imbalance, microthrombi, and hypoperfusion are incriminated. Subendocardial hemorrhagic necrosis was present in 17 of 40 patients whose postoperative survivals were four days or less. Its frequency increased with duration of survival. Hypoperfusion of the myocardium is suggested as the major etiologic factor of the subendocardial hemorrhagic necrosis. Myocardial lesions observed among these 50 patients did not differ from those in patients who had open-heart surgery for other reasons.

Aortic dissection complicating aortocoronary saphenous vein bypass.

A high autopsy incidence, 7 of 50 cases (14%), of aortic dissection as the major cause of death in patients who died following aortocoronary saphenous vein bypass is reported. The patients ranged in age from 45 to 61 years, and all had moderately severe aortic atherosclerosis. None was hypertensive, and no significant predisposing aortic medial disease was recognized. Trauma during surgical cannulation with retrograde perfusion through atherosclerotic femoral or iliac arteries was considered the initiating event leading to ileo-aortic dissection in five of the seven patients. Damage secondary to aortic cross-clamping is suggested as the pathogenetic mechanism leading to dissection in the other two patients. In six patients, the dissection was Type I of DeBakey, and in one patient, Type II. Dissection was recognized intra-operatively in five of the seven patients. In older patients, particularly those likely to have atherosclerosis of the aorta and its main branches, vigilance regarding the possibility of aortic dissection developing during the course of cardiopulmonary bypass must be constant.

Prostatic origin of tumors. An immunohistochemical study.

An immunoperoxidase technic was used to localize prostatic acid phosphatase in a variety of primary and metastatic neoplasms. The aim was to explore the histogenesis of tumors affecting the prostate gland and to demonstrate the prostatic origin of metastases in various sites. A highly specific antiserum to prostatic acid phosphatase was raised in rabbits, and the peroxidase-antiperoxidase procedure was carried out on formalin-fixed paraffin-embedded routine pathology material. All specimens from the 37 cases of known primary and metastatic prostatic carcinomas stained positively for prostatic acid phosphatase, regardless of their histologic differentiation. None of the specimens from the 44 cases of proven nonprostatic primary and metastatic tumors stained positively for prostatic specific acid phosphatase. The data suggest that demonstration of prostatic acid phosphatase by the immunoperoxidase technic is a practical, sensitive, and specific test for the prostatic origin of an otherwise unclassifiable primary or metastatic neoplasm.

Immunohistochemical markers for prostatic cancer.

In a patient with an unclassifiable primary or metastatic neoplasm, with or without a history of prostatic cancer, immunostaining for PA or PSAP may prove invaluable. The procedure is simple, rapid, inexpensive, and extremely accurate in demonstrating the prostatic origin of tumors. It should be noted however, that the specificity of results is entirely dependent upon the specificity of the primary antibody, which should be meticulously defined before the procedure is used for diagnostic purposes.

Detection of p53 gene abnormality by sequence analysis of archival paraffin tissue. A comparison with fresh-frozen specimens.

This parallel study was designed to compare the sensitivity and specificity of detection of point mutations in fresh-frozen and formalin-fixed, paraffin-embedded breast cancer tissue. Sequence analysis of exon 5 of p53 gene was performed on polymerase chain reaction-amplified DNA from 25 infiltrating ductal carcinomas of the breast. Four tumor showed mutations with identical base substitutions in their respective codons of both frozen and paraffin-embedded specimens. We conclude that subtle genetic alterations can be detected in archival paraffin tissue with an accuracy comparable to that of fresh-frozen histologic samples.

Prekeratin in spindle cell tumors of the skin.

A variety of benign and malignant skin lesions was stained for the presence of prekeratin using an unlabeled antibody peroxidase-antiperoxidase method and an antibody raised against human prekeratin protein. No prekeratin could be detected in benign or malignant lesions derived from melanocytes, and prekeratin could not be found in atypical fibroxanthoma, Kaposi's sarcoma, angiosarcoma, dermatofibrosarcoma protruberans, and leiomyosarcoma. Paget's cells did not contain prekeratin. Varying staining intensities were observed in keratinocytic atypical hyperplasias. Of particular interest is the observation that many spindle cell lesions that were difficult to identify with 100% assurance from routine stains could be positively identified as squamous cell carcinoma after prekeratin was found in the atypical cells.

Multiple mutations of the p53 gene in human mammary carcinoma.

Alteration of the p53 tumor suppressor gene is the most common genetic abnormality in human cancer. In breast cancer, depending on the stage of disease and method of detection, mutation rates of 25-60% have been observed. Multiple mutations of p53 gene in the same tumor however, are rarely reported. In this study we explored the frequency of multiple mutations of p53 gene in mammary carcinoma in a cohort of south Florida patients. Three hundred eighty-four cases of primary breast cancer diagnosed between 1984 and 1986 at the University of Miami, Jackson Medical Center were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on cloned PCR-amplified DNA of formalin-fixed, paraffin-embedded tumors. Two hundred thirty-four of 384 breast cancers (61%) had p53 mutation. Of those, 36 tumors showed more than one mutation; 31 tumors had two mutations, three showed three, one tumor had five mutations, and one case carried six mutations. The majority of mutations were missense (43) followed by silent (35); and most occurred within a single exon. Our study suggests that multiple mutations of p53 suppressor gene in breast cancer are more common than currently believed.

Maximizing the effectiveness of the autopsy in cases of sudden death.

Autopsies are essential in the investigation of sudden unexpected deaths. To maximize the effectiveness of the autopsy in these cases, the pathologist must assess the circumstances leading to the person's death, should be knowledgeable of the diseases most often responsible for sudden death, and should use dissection techniques most appropriate for their disclosure. In adults, the great majority of sudden deaths are cardiogenic and related to diseases of the coronary arteries, cardiac valves, or myocardium. In infants and young children, however, extracardiac diseases are prevalent in the causation of sudden deaths. Approximately one third of these are inexplicable (sudden infant death syndrome).

Prekeratin immunohistochemistry in the diagnosis of undifferentiated carcinoma of the nasopharyngeal type.

We investigated the value of prekeratin immunostaining in establishing the diagnosis of undifferentiated carcinoma of the nasopharyngeal type (lymphoepithelioma). As in squamous cell carcinoma of the nasopharynx, all seven lymphoepithelial carcinomas stained for prekeratin whereas other look-alike but histogenetically different neoplasms (malignant lymphoma, esthesioneuroblastoma, and small round cell sarcomas) did not stain. In addition to confirming the squamous epithelial nature of lymphoepithelioma, our findings indicated that immunoperoxidase staining for prekeratin is a useful diagnostic tool in the differential diagnosis of nasopharyngeal carcinomas in general and lymphoepithelioma in particular.