A method for measuring cyto-adherence of frozen tissue sections

A simple method for quantifying the cells adhering to frozen sections of mouse spleen using a microscope equipped with a drawing tube attachment, and a planimeter is described. It is a straightforward and reproducible technique which has been preliminary employed to study optimal binding conditions for E_sheep and E_oxAC.     

Ultrarapid Ki-67 immunostaining in frozen section interpretation of gliomas

BACKGROUND: Astrocytic tumours, the most common gliomas, are often classified intraoperatively using standard morphological staining. The final diagnosis and grading of gliomas on paraffin wax sections is often assisted by Ki-67 immunohistochemistry, but standard immunostaining protocols take too long to be used intraoperatively. AIMS: To investigate a new rapid Ki-67 immunohistochemical test for its use in an intraoperative setting. METHODS: The new Ki-67 immunostaining (Ultrarapid-Ki67) method on frozen sections can be carried out in 10 minutes. Thirty four pilocytic and diffuse astrocytomas were immunostained by rapid Ki-67 and results were compared with corresponding MIB-1 staining, histological grading, and prognosis. RESULTS: The staining protocol was practical to perform and the results were morphologically and quantitatively indistinguishable from those after immunostaining with MIB-1, an antibody recognising Ki-67 in paraffin wax embedded tissue. A comparison of Ultrarapid-Ki67 and MIB-1 immunostaining of paraffin wax sections showed almost identical quantitative correlation in astrocytic gliomas (r = 0.916; p<0.001). The Ultrarapid-Ki67 indices (percentage of positive cells) of low grade (I/II) astrocytomas ranged from 0% to 6.1%, whereas those of representative high grade (III/IV) tumours were significantly higher (range, 5.6-45%; p<0.001). The best prognostic cutoff point for Ultrarapid-Ki67 was 7.5%, which divided diffuse grade II-IV astrocytomas into significantly differing subsets (p = 0.0008). CONCLUSION: Ultrarapid-Ki67 immunostaining is a useful adjunct to morphological diagnosis and grading of astrocytic tumours, and as a fast test (approximately 10 minutes for staining plus three to four minutes for scoring), it could be used in routine intraoperative diagnosis of gliomas and other neoplastic diseases.     

Current practices in performing frozen sections for thyroid and parathyroid pathology

In this review article, current trends in thyroid and parathyroid frozen sections are discussed. In Japan and other countries, the numbers of thyroid frozen sections have been dramatically decreasing over the past decade. The decline in the number of thyroid frozen sections has been attributed to two major factors: highly diagnostic preoperative fine needle aspiration for papillary carcinomas, the most frequent type of thyroid cancers, and the acknowledgment in the literature of the disadvantageous frozen sections for follicular tumors. Several authors have argued that the frozen section of thyroid nodules should be limited only to cases that have a preoperative cytology diagnosis as "atypical" or "suspicious". In contrast, frozen sections for parathyroid glands have been increasing in numbers. This increase is thought to be largely due to the high number of parathyroidectomies for secondary hyperplasia in dialysis patients. Frozen sections are usually performed to confirm the removal of parathyroid tissue for either cyropreservation or auto-transplantation. It is concluded that thyroid and parathyroid frozen section examination is restricted to selected situations.     

New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections.

AIMS--To prepare a rabbit antiserum equivalent to MIB 1 to permit the simultaneous assessment of cell proliferation and other markers of interest using double labelling studies. METHODS--Rabbits were immunised with a synthetic peptide deduced from the cDNA sequence coding for the Ki-67 antigen. Serum samples were tested for immunoreactivity using different immunobiochemical methods. RESULTS--A polyclonal antiserum was derived which detects the native as well as recombinant parts of the Ki-67 antigen in different test systems. Furthermore, the antiserum stains the Ki-67 antigen in routinely processed, paraffin wax embedded material. CONCLUSIONS--After antigen unmasking by microwave treatment the antiserum described here represents a powerful tool for the determination of growth fractions even in archival material. It is especially suitable for double staining experiments in combination with monoclonal antibodies.     

A prospective trial of telepathology for intraoperative consultation (frozen sections)

Telepathology is a maturing technology that, for a variety of reasons, has not been widely deployed. In addition, clinical validation is relatively modest compared with accepted telemedicine applications such as teleradiology. A prototype telepathology system (Tele-Path(sm)) featuring high-resolution images selected from a remote microscope site has been developed at the University of Alabama at Birmingham (UAB). To validate the diagnostic efficacy of the system, a prospective study was undertaken of parallel diagnoses by conventional microscopy and telepathology with a remotely operated microscope. Slides from 99 intraoperative consultations from 29 tissue/ organ sites in the University of Alabama Hospitals by 9 academic pathologists were used in the study. Each microscopic and telepathology diagnosis was compared with the final diagnosis rendered by a referee pathologist. Diagnoses were classified as correct, false positive, or false negative or classification error. Of the 99 frozen sections evaluated, 3 cases were deferred. Of the remaining 96 cases, 2 received incorrect diagnoses in both the microscopic and telepathology arms of the study. Three errors occurred only in the telepathology arm. There was 1 false-positive diagnosis, 1 false-negative diagnosis, and 1 classification error. Statistical analysis indicated no significant difference between telepathology and conventional microscopy. Qualitative data indicated that the pathologists were generally satisfied with the performance of the system. Telepathology using this system paradigm is sufficiently accurate for real time utilization in a complex surgical environment. Telepathology therefore may be an effective model to support the surgical services of hospitals lacking full-time pathology coverage, resulting in full-time access to anatomic pathology services.     

Improved method for immunoperoxidase detection of membrane antigens in frozen sections.

The visualisation of membrane antigens in frozen sections using metal-enhanced 3,3'diaminobenzidine (DAB) was investigated. Particular attention was paid to the degree of reaction enhancement, the disruptive effect on morphological detail, and the ease of the techniques. The best result was obtained using nickel-modified DAB at pH 6.0 to develop the peroxidase enzyme, with further enhancement in cobalt chloride at neutral pH. Silver methenamine enhancement is also possible but can give rise to non-specific or background staining.     

A rapid technique of preparing frozen sections of small brains

The preparation of cryostat sections of unfixed tissue for use in evaluating the extent of brain lesions is described. The method is applicable to mouse, rat, rabbit and other small brains.     

Accuracy of and reasons for frozen sections: a correlative, retrospective study

Previous studies on the accuracy of frozen sections (FS) were not based on the reasons for which the FS was performed. By omitting this important information, those studies reported FS accuracy of 94% to 97.4%. The current study analyzes the accuracy of FS diagnoses based on the reasons for which they were conducted. Of 482 specimens examined by FS in 1986, 41 were performed for evaluation of section margins of tumors, 29 for identification of unknown tissue, and 43 for detection of lymph node metastases. All 113 of these examinations proved to be 100% accurate. The remaining 369 FS were performed for diagnosis of an unknown pathologic process. Of these, 83.47% were precisely diagnosed; in 10.30%, the pathologic process was correctly, but not precisely, diagnosed; in 3.79%, the diagnosis was deferred; and the remaining 2.44% were incorrectly diagnosed (with no harmful consequences to the patients). By eliminating the cases of deferred diagnoses, the accuracy rate increased to 86.76%. Therefore, we suggest that the pathologist and the surgeon should not draw any conclusion from an inconclusive interpretation of FS and should proceed as though an FS had not been performed. When combining results of "precise diagnoses" with those of "correct pathologic process," the overall accuracy rate increased to 97%. The investigators conclude that FS should be used for a general diagnosis of a pathologic process rather than for an exact or precise diagnosis. Finally, we suggest that a similar survey of FS be periodically performed in every pathology department as part of its quality assurance program.     

Electron microscopy of virus particles in ultrathin frozen sections.

A procedure with the use of polyethylene glycol (PEG) 600 to support biological materials for ultracryotomy was developed. Developing and mature virions were demonstrated by electron microscopy in negatively stained frozen sections of vaccinia virus-infected human diploid cells and of herpes simplex virus (HSV) type 1 infected primary rabbit kidney cells. Approximate localisation of the virus particles in the cells was possible.