Automated molecular genetic DNA analysis for detecting B-cell non-Hodgkin''s lymphoma in cytologic specimens

The effect of a calpain-selective cell permeant inhibitor, benzyloxycarbonyl Leu-Leu-Tyr diazomethylketone (ZLLY-CHN2), on the serum-stimulated growth of WI-38 human fibroblasts has been investigated. Only cell permeant protease inhibitors with activity against calpains prevented progression into S-phase. Protein blotting experiments indicated that p53 immunoreactivity increased in late G1 cells treated with ZLLY-CHN2. The content of p21Waf1/Cip1 CDK inhibitor also increased, providing a mechanism for the observed failure to enter S-phase. Further studies indicated that p53 could be degraded by a ZLLY-CHN2-sensitive protease immediately prior to S-phase, but that proteolysis did not occur after this critical time point. Chelation of extracellular Ca2+ by addition of EGTA inhibited the p53 degradation. Consistent with proteolysis of p53 in late G1 phase, mu-calpain immunoreactivity transiently accumulated in cell nuclei at this time. ZLLY-CHN2 did not appear to increase p53 mRNA in WI-38 cells. Purified mu-calpain required only 1 to 3 microM Ca2+ to proteolyze p53 in WI-38 cell lysates. These results indicate that ZLLY-CHN2 inhibits progression of WI-38 cells into S-phase by inactivating a calpain-like protease that is responsible for proteolysis of constitutively expressed p53 in late G1.     

Presence of Streptococcus DNA sequence in surgical specimens of gastric cancer

In Southern blot analysis using Mycoplasma 16S ribosomal DNA (rDNA) as a probe, positive signals were detected in DNA samples from surgical specimens of gastric cancers. The DNA that hybridized to Mycoplasma 16S rDNA was eluted from the gel, cloned and sequenced. The cloned sequence was identical to 16S rDNA of Streptococcus anginosus. In Southern blot analysis with the S. anginosus 16S rDNA fragment as a new probe, positive signals were detected in 9 (20%) out of 43 cases of gastric cancer.     

Analysis of 24,444 surgical specimens accessioned over 55 years in an ophthalmic pathology laboratory

PURPOSE: To summarize the pathologic diagnoses of a large number of surgically-obtained specimens over an extended time period in a single ophthalmic pathology laboratory. METHODS: We analyzed the records of 24,444 surgically obtained specimens accessioned in the L.F. Montgomery Ophthalmic Pathology Laboratory, Emory University, Atlanta, GA between May 1941 and December 1995. Age, sex, topography, clinical procedure, and histologic diagnosis were entered into a database using the modified SNOMED coding system. The diagnosis of the surgically enucleated eyes were analyzed with respect to years of enucleation. RESULTS: The most common topographic area associated with a histologic diagnosis was the cornea (39.3%), followed by lens (16.0%), vitreous (12.0%), uvea (9.8%), eyelids (8.0%), conjunctiva (7.7%), retina (7.7%), and orbit (2.1%). The relative proportion of vitreous specimens has continuously increased and became the most common surgical specimen in 1995. The most common underlying disease of surgically enucleated eyes is trauma (40.9%), followed by ocular neoplasia (24.2%), 'surgical' diseases of the cornea, lens and retina including glaucoma (17.3%), vascular diseases (6.7%), and inflammatory conditions (6.7%). The relative frequency of trauma and ocular inflammation as a cause of enucleation decreased significantly (p < 0.05) over the time of the study period while the relative proportion of ocular neoplastic processes increased (p < 0.0001). CONCLUSIONS: The availability of new surgical techniques has caused a change in the relative frequencies of different ocular specimens submitted for histologic examination.     

Flow cytometric DNA measurements in aspiration biopsies and surgical specimens of breast cancer

OBJECTIVE: One of the prognostic factors in breast cancer is the proliferation activity of the tumor. This study sought knowledge of this activity before surgery to benefit the design and timing of therapy. STUDY DESIGN: Flow cytometric DNA analysis data from 52 diagnostic fine needle aspirates were compared with data from subsequent surgical specimens. RESULTS: The data showed that the coefficient of variation of the G1 peak was lower in the aspirates. Small, near-diploid peaks were detected more frequently in aspirate histograms than in surgical specimens. DNA analyses by flow cytometry from aspirates, which can be obtained prior to surgical treatment, were as reliable as those obtained from surgical specimens, provided that the cellular material was diagnostic of cancer. CONCLUSION: Our results suggest that flow cytometry DNA analysis from the first preoperative cytologic specimen from a breast tumor will permit faster planning and coordination of breast cancer care.     

Multiparametric in situ messenger RNA hybridization analysis to detect metastasis-related genes in surgical specimens of human colon carcinomas

We examined the expression of several genes that regulate different steps of metastasis in surgical specimens of human colon carcinomas. The expression of epidermal growth factor receptor (growth), basic fibroblast growth factor [(bFGF), angiogenesis], type IV collagenase (invasion), E-cadherin (adhesion), and multidrug-resistant (mdr)-1 (drug resistance) mRNA was examined using an in situ mRNA hybridization (ISH) technique and Northern blot analysis. Dukes' stage C and D tumors exhibited a higher level of expression (P <0.05) for bFGF, type IV collagenase, and mdr-1 mRNA than Dukes' stage B tumors. The expression level of epidermal growth factor receptor and E-cadherin did not correlate with the stage of the disease. The ISH technique revealed intertumoral heterogeneity for expression of several genes among Dukes' stage B neoplasms. In some Dukes' stage B tumors, we also found intratumoral heterogeneous staining for bFGF and type IV collagenase, with the highest expression level at their invasive edge. In Dukes' stage C and D tumors, the expression of these genes was more uniform. These results recommend the suitability of the multiparametric ISH analysis for metastasis-related genes to identify individual colon cancers with metastatic potential.     

Value of fluorescence in situ hybridization for detecting the bcr/abl gene fusion in interphase cells of routine bone marrow specimens

A patient presented with a severe nephrotic syndrome and a renal biopsy consistent with early focal segmental glomerulosclerosis (FSGS). After a year of intensive immunosuppressive therapy proteinuria was unabated and renal function began to deteriorate. Treatment with weekly plasmapheresis combined with moderate doses of prednisone and azathioprine produced a dramatic decrease in proteinuria and serum creatinine. A marked fall in the B-cell population occurred during treatment, as well as an increase both in T-lymphocytes of the mature CD4+ helper/suppressor phenotype and in the immature/cytotoxic CD8+ phenotype. Activation of the immune system during treatment was demonstrated by an increase in the rate of spontaneous proliferation of peripheral blood mononuclear cells and an increase in T-cell expression of the interleukin-2 receptor.     

Departmental audit in surgical anatomical pathology

Chronic obstructive pulmonary disease (COPD) is the result of many years of accelerated decline in lung function in susceptible cigarette smokers. Although risk factors for the susceptibility of smokers to COPD have been established, there are still large gaps in our knowledge of the biological basis for these risk factors and of how to identify individuals at risk. COPD is the fourth leading cause of death and, in contrast to other major chronic diseases in the United States, has not shown declines in mortality over the past 20 years. Mortality trends reflect patterns of initiation of cigarette smoking that occurred 30 to 50 years previously. Current mortality trends indicate that COPD mortality may be leveling off among white males, but will continue to increase among women, African-Americans, and the elderly. Recent studies indicate that early identification of individuals with airflow obstruction and smoking intervention can halt the progression of COPD, but widespread screening and intervention programs have not yet been established.     

Failure to detect the presence of Chlamydia pneumoniae in sarcoid pathology specimens

The pathogenesis of sarcoidosis is not yet known. On the basis of seroepidemiological data, an association between Chlamydia pneumoniae infection and sarcoidosis has been suggested, but so far no study has addressed the direct detection of this agent in the affected tissues. The aim of the present study was to detect C. pneumoniae deoxyribonucleic acid (DNA) within sarcoid tissue specimens by means of a two-step polymerase chain reaction. Lung biopsy specimens of 33 patients with histologically confirmed pulmonary sarcoidosis and 21 control lung biopsies or pathology specimens of patients with pulmonary carcinoma or emphysema were retrospectively analysed. A nested polymerase chain reaction was applied using two sets of primers designed to detect a fragment of the 16 strand ribosomal ribonucleic acid (rRNA) gene of C. pneumoniae. The results of the study failed to demonstrate the presence of C. pneumoniae in biopsy specimens of sarcoid tissue and in the control lung biopsies or pathology specimens. Our results, therefore, tend to rule out the possibility of a direct involvement of Chlamydia pneumoniae in the pathogenesis of sarcoidosis.     

The surgical pathology of the appendix in South African blacks

A case of deep dermatophytosis in the gluteal region in a male patient successfully treated with terbinafine is described with its clinical, mycological and histopathological features.     

Centrosome defects and genetic instability in malignant tumors

Genetic instability is a common feature of many human cancers. This condition is frequently characterized by an abnormal number of chromosomes, although little is known about the mechanism that generates this altered genetic state. One possibility is that chromosomes are missegregated during mitosis due to the assembly of dysfunctional mitotic spindles. Because centrosomes are involved in spindle assembly, they could contribute to chromosome missegregation through the organization of aberrant spindles. As an initial test of this idea, we examined malignant tumors for centrosome abnormalities using antibodies to the centrosome protein pericentrin. We found that centrosomes in nearly all tumors and tumor-derived cell lines were atypical in shape, size, and composition and were often present in multiple copies. In addition, virtually all pericentrin-staining structures in tumor cells nucleated microtubules, and they participated in formation of disorganized mitotic spindles, upon which chromosomes were missegregated. All tumor cell lines had both centrosome defects and abnormal chromosome numbers, whereas neither was observed in nontumor cells. These results indicate that centrosome defects are a common feature of malignant tumors and suggest that they may contribute to genetic instability in cancer.     

Diagnosis of genetic defects by chromosomal analysis

Of 901 karyotypes performed over a period of 4 years, genetic anomalies were detected in 162 cases. Down's syndrome (trisomy 21) was the most common (168.8%) genetic disorder followed by Turner's syndrome, Philadelphia chromosome, Klinefelter's syndrome, Edward's syndrome (trisomy 18) and Patau's syndrome (trisomy 13). All the three trisomies were detected very early in life. Mean age at the time of diagnosis for Turner's syndrome was 13.3 years, allowing a timely hormone replacement therapy to improve secondary sexual characters. Patients with Klinefelter's syndrome were diagnosed late (mean age 23.6 years), which greatly reduced their chances of an effective therapy to improve the clinical and social outcome.     

Monoclonal antibodies detecting components of the bovine immune system in formaldehyde-fixed paraffin-embedded tissue specimens

Although the use of monoclonal antibodies in the diagnosis of fixed human material is a daily routine, the lack of suitable reagents recognizing epitopes resistant to formaldehyde fixation is an obvious limit to extending this approach to veterinary research and practice. To find reagents that retain their binding capacity to the recognized epitopes in formaldehyde-fixed samples, bovine lymphoid tissue sections were immunostained using various antigen retrieval procedures with monoclonal antibodies raised against ruminant leukocyte cell surface molecules. As a results, a set of antibodies could be established that allowed the identification of different immune cell types including all or a distinct subpopulation of B and T lymphocytes, monocytes, macrophages, granulocytes, red blood cells, and vascular endothelial cells.     

地质雷达在拦渣坝体隐蔽缺陷探测中的应用

地质雷达是一种新发展起来的物探方法,在很多行业得到广泛应用。介绍了地质雷达工作原理和探测工程实例,通过地质雷达的实测成果和图像分析,说明该项新技术在探测坝体内部隐蔽缺陷中的良好效果。工程实际应用表明,实施地质雷达探测费用较低,效果好,经济效益显著,是一种值得推广、有广阔应用前景的物探方法。     

Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens

The rapid identification of mycobacterial DNA in clinical samples by PCR can be useful in the diagnosis of tuberculous infections, but several large studies have found that the sensitivity of this approach is not better than that of culture. In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients with paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other potential inhibitors of the amplification reaction (sequence capture-PCR). By using this approach to capture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat region), it was possible to detect as little as one genome of mycobacterial DNA in samples containing up to 750 micrograms of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification. Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleurisy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negative samples. In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the three culture-positive samples were positive by PCR. The sensitivity of detection of the direct repeat sequence in these samples by sequence capture-PCR was similar to that of IS6110 and, in addition, permitted immediate typing of the strains from some patients. We conclude that sequence capture-PCR improves the sensitivity of detection of mycobacterial DNA in paucibacillary samples. This approach should be useful in detecting rare target sequences from organisms implicated in other pathologic processes.     

Frozen-section diagnosis in surgical pathology: a quality assurance study

In a quality assurance study we reviewed one thousand four hundred and forty-three consecutive frozen sections performed at department of pathology, VGH-TC from June 1995 to July 1996. The diagnostic accuracy was 92.6%. The diagnosis was deferred in sixty-eight cases (4.7%). False positive for malignant tumor was made in two cases (0.14%) and false negative diagnosis for malignancy in thirty-seven (2.56%). The inaccurate diagnosis was mainly in samples taken from the brain, female breast, and thyroid. Incorrect diagnoses were mainly due to interpretation of the pathologic findings (71.8%), followed by gross sampling (15.4%) and microscopic sampling (12.8 %). Some of the lesions were difficult to diagnose even in permanent sections. Technical skill and diagnostic expertise are essential for frozen diagnosis. We suggest that an accuracy survey of frozen section be periodically performed in every pathology department as part of its quality assurance program.