Localized in situ amplification (LISA): a novel approach to in situ PCR.

Amplification of specific gene target sequences has become a routine molecular procedure in a variety of laboratories. When coupled with either a direct or indirect method of detecting amplified product, in situ amplification offers an extremely powerful investigative tool. We describe a protocol for a localized in situ amplification (LISA) reaction that includes tissue-culture cloning rings and allows for the amplification of gene target sequences in specific regions of paraffin-embedded tissue sections. Digoxigenin-11-dUTP was added to the amplification reaction and thus incorporated into the amplified products, providing a mechanism by which direct nonisotopic detection could be performed. To demonstrate the approach, LISA was performed on known positive Pneumocystis carinii rat lung tissues, with primers specific for the P. carinii rRNA gene sequence.

Hypersegmentation of neutrophils in the cerebrospinal fluid: report of a case with hematologic correlation and review of the literature.

Hypersegmented neutrophils can occasionally be seen in the cerebrospinal fluid (CSF) of patients with acute pyogenic meningitis, and rarely as an artifact of cytocentrifugation. We report a case of florid neutrophil hypersegmentation in cytocentrifuged CSF specimens from a patient presenting with anemia and mental status changes. Megaloblastic anemia was excluded by a thorough hematologic evaluation, and the patient was later found to have an epidural abscess. The striking hypersegmentation is attributed to the combined effects of meningeal infection and cytocentrifugation, and a review of the available limited literature on cytomorphologic alterations in CSF cells, specifically neutrophils, is presented.

Prognostic factors in the recurrence of stage I and II squamous cell cancer of the oral cavity.

Stage I and II squamous cell cancers of the oral cavity have a high recurrence rate given their size and relative amenability to surgical resection. It has been suggested that one way to decrease this recurrence rate is to augment the surgical resection of these tumors with either elective neck dissection or radiation therapy. However, this would expose a significant number of patients to the unnecessary morbidity associated with either of these modalities. In an attempt to identify those patients most at risk for recurrence, we retrospectively determined the clinical and histologic factors that were associated with recurrence in 49 patients with stage I and II oral cavity cancer. Multiple regression analysis revealed that when various interactions between variables were controlled for, only the presence of a positive surgical margin or a tumor depth greater than 5 mm was significantly associated with recurrence. Each individually increased the likelihood of recurrence almost threefold.

The changing trends of uterine cancer and cytology: a study of morbidity and mortality trends over a twenty year period.

Data on squamous carcinoma of the cervix from a 20 year study period (1955 to 1974) in metropolitan Toledo revealed a 66% reduction of the average annual age-adjusted incidence rate and a 61% reduction in death rate of cervical squamous carcinoma when the first time period (1955 to 1958) was compared with the last time period (1971 to 1974). The decrease for both morbidity and mortality rates was more pronounced in women age 50 years and younger. The age-adjusted death rate during this study period revealed 15.5/100,000 for black women and 8.7/100,000 for white women. The reduction in death rate of 83% in black women is more prominent than 54.5% in white women. The decrease in both morbidity and mortality for cervical squamous carcinoma has a close relation to cytologic screening activity. The factors of age and race, probably related to socioeconomic status, are two known determinants of risk for cervical squamous carcinoma. Data for endometrial carcinoma during this study period revealed 15.5/100,000 women in the average age-adjusted incidence and an increase of 13.8% in average yearly rates when the first time period (1955 to 1958) were compared with the last time period (1971 to 1974). The peak age was 60 to 64 years old in the first time period and shifted to 70 to 74 years old in the last time period. The trend in metropolitan Toledo is comparable to that of Louisville, Kentucky. The mass cytologic screening program which contributed to a remarkable reduction in morbidity and mortality for the cervical squamous carcinoma, did not have any beneficial effect on endometrial carcinoma.

An acceptable yearly screening device for endometrial carcinoma.

PIP: Endometrial carcinoma is a disease of menopausal and postmenopausal women with 74% of cases occurring between the ages of 50-69 years and 16% at age 70 or older. Recent reports suggest an increase in the frequency of the lesion and occurrence at an earlier age. Any single test by most of the conventional methods will miss about 50% of the carcinomas of the endometrium. To obtain greater accuracy in diagnosis, it is necessary to obtain large numbers of well-preserved cells directly from the endometrial cavity. Cervical patency should be proved in the examination of all postmenopausal women. The device described uses a silicone rubber tube 33.5 cm in length and 2 mm in diameter. A wire stylet for stiffness and a rounded plastic tip facilitate passage through the sometimes stenotic cervical canal. Near the tip many small holes permit aspiration of endocervical secretions. A plastic collar around the tubing facilitates the aspiration. An aspirating syringe adapter is needed. After conventional Pap smears are obtained the aspirating tube is passed into the uterus. Little or no pain is produced. Negative pressure for aspiration is obtained with a syringe. The contents of the aspirating tube are expelled onto a slide. The slide preparations are immediately fixed in a bottle of absolute alcohol containing 3% glacial acetic acid. The acid lyses most red blood cells. This method was used in 983 outpatients and 500 hospitalized patients. There were only 5 patients in whom the endometrial aspirator could not be passed, due to previous cervical trauma in 3 and vaginal atrophy and distortion in 2. No uterine perforations or infections occurred. There were no positive tests for carcinoma in the 983 outpatients. Endometrial carcinoma was diagnosed histologically in 32 of the 500 hospitalized patients. IN 27 of these 32 cases the endometrial carcinoma had been detected by cytological examination of the aspirate. There were 5 carcinoma cases in which the aspirate did not show malignant cells. In 3 of them external radiation had been given prior to surgery and some residual cancer was found in the hysterectomy specimen. Excessive blood on the slide had prevented adequate cytologic evaluation in the other 2. In 6 cases adenocarcinoma was suspected from the examination of the aspirate but not found histologically. 4 of them showed abnormal endometrial curettings. Polyps were found in 3 and hyperplasia in 1. 8 cases of in situ or early invasive carcinoma of the cervix were discovered. Results show that the method is safe and accurate to an 84.4% degree for detecting endometrial adenocarcinoma. In an accompanying discussion, better fixation of material on slides was suggested. Annual mass population screening by this method was not considered practical. Others have depended on a jet wash of the endometrium and on endometrial biopsies to obtain material for histologic study, but many patients do not like having the needed dilatation and fail to return for reexaminations.^ieng