Intraoperative cytologic diagnosis of sentinel node metastases in breast cancer

OBJECTIVE: To clarify the usefulness of imprint cytology for intraoperative investigations of sentinel lymph nodes in breast cancer, comparing the results with those of examinations using frozen and permanent sections. STUDY DESIGN: The material consisted of 303 sentinel lymph nodes from 124 cases of clinically node negative breast cancer. Touch imprint cytologic slides and frozen sections were obtained from the same cut surface of the sentinel nodes. Correlations with the final histopathologic results in paraffin sections were evaluated. RESULTS: The sensitivity, specificity and accuracy of imprint cytology were 70.3%, 99.6% and 96.0%, and those of frozen sections were 83.8%, 100%, 98.0%, respectively. The values were improved when the 2 methods were combined (89.2%, 99.6%, 98.3%), though the concordance between imprint cytology and frozen section was 91.9%. CONCLUSION: Both imprint cytology and frozen section are useful for evaluating sentinel lymph node status in breast cancer. However, the 2 techniques should be combined to improve the diagnostic sensitivity.     

Intraoperative evaluation of sentinel lymph nodes in breast cancer: comparison of frozen sections,imprint cytology and immunocytochemistry

M. Francz, K. Egervari and Z. Szollosi
Intraoperative evaluation of sentinel lymph nodes in breast cancer: comparison of frozen sections, imprint cytology and immunocytochemistry Objective: We analysed the utility of imprint cytology with rapid immunocytochemistry and frozen section analysis for the evaluation of sentinel lymph nodes in breast cancer patients. Methods: The sensitivity, specificity, and positive and negative predictive values have been calculated for each method individually, each pair and all three together. We compared these results with those of routinely processed paraffin sections. Results: The sensitivity and specificity of each of the three methods for detection of metastatic carcinoma were as follows: 69.4% and 97.8% for touch imprint cytology; 58.3% and 100% for frozen sections; 68.5% and 98.9% for rapid immunocytochemistry. When the methods were combined, the highest accuracy was achieved by touch imprint cytology, frozen sections, touch imprint cytology plus rapid immunocytochemistry, or touch imprint cytology frozen section analysis and rapid immunocytochemistry, each of these having identical sensitivity and specificity of 72.2% and 97.8%, respectively. Conclusions: In our study the combined accuracy of the three methods was the same as combining touch imprint cytology and frozen sections or touch imprint cytology plus rapid immunocytochemistry. Rapid immunocytochemistry provides an additional parameter and preserves tissue for permanent sections.     

Role of exfoliative cytology in diagnosis of laryngeal tumors

OBJECTIVE: To evaluate the value of cytologic examination in the diagnosis of laryngeal tumors. STUDY DESIGN: A series of 100 patients with suspected tumorous lesions of the larynx were included in the study. The material for cytologic analysis was obtained by use of a modified instrument, that is, a slightly concave discoid knife, during direct microlaryngoscopy, followed by biopsy for paraffin and frozen section histopathology. The findings obtained by cytology and frozen section histopathology were compared with the findings of paraffin section histopathology. RESULTS: Cytology showed an 83.9% sensitivity, 100% specificity and 90% accuracy compared with histopathologic findings (p < 0.05; chi2 = 7.7). Frozen section pathohistology vs. paraffin section histopathology showed an 80.6% sensitivity, 97.4% specificity and 87% accuracy (p < 0.05; chi2 = 8.1). CONCLUSION: Cytologic examination showed higher accuracy compared to frozen section histopathology, also giving an insight into the type of laryngeal pathology in the case of precancerous lesions. The use of cytology and frozen section histopathology during direct microlaryngoscopy improves clinical diagnostic accuracy.     

Urothelial neoplasms of the upper urinary tract. A correlation between cytologic and histologic findings in 43 patients with urothelial neoplasms of the renal pelvis or ureter.

The present study elucidates the possibilities to diagnose and classify urothelial tumors of the upper urinary tract by means of exfoliative cytology on voided urine using a membrane filter method. In a series of 30 patients with renal pelvic tumors and 13 patients with ureteral tumors an overall agreement between cytology and histopathology was obtained in 25 cases (58%). None of the Grade 1 tumors or of the non-invasive Grade 2 tumors were regarded as positive by cytology whilst two out of five invasive Grade 2 tumors had positive cytologic reports. The series included 24 patients with poorly differentiated or anaplastic tumors, 17 of whom had positive cytology (71%). By excluding from the series 13 patients with obstructed urinary passages or radiologically non-functioning kidneys on the tumor side an agreement between cytology and pathology was reached in 83 per cent of the cases, regardless of tumor grade, and in 17 out of 18 patients with Grade 3-4 tumors (94%).     

Diagnostic accuracy and role of immediate interpretation of fine needle aspiration biopsy specimens from various sites

A statistical analysis of the accuracy of the immediate interpretation of 425 fine needle aspiration (FNA) biopsies from various sites was performed. Preliminary interpretation of Diff-Quik-stained smears was rendered within a few minutes after performing the FNA biopsy, using diagnostic terminology similar to that of a surgical pathology report. The immediate assessment was documented in the formal cytology report and compared to the final diagnosis. For the entire series, the immediate interpretation had a sensitivity of 96%, a specificity of 97%, a positive predictive value (PV) of 98%, a negative PV of 95% and an efficiency of the test of 96%. There were 14 false-negative or falsely insufficient immediate interpretations and one false-positive immediate diagnosis. The diagnostic accuracy of the immediate interpretation of FNA biopsies from specific sites was also calculated; FNA biopsies of the pancreas were the least accurate procedure, having a sensitivity of 60% but a specificity of 100%. The role of the immediate interpretation of FNA biopsies is similar to the use of frozen sections in surgical pathology. An immediate assessment can (1) determine whether an adequate specimen is present, (2) render a specific preliminary diagnosis, (3) guide further clinical investigations or treatment, and (4) determine whether ancillary studies are needed to make a more accurate or specific diagnosis from the FNA specimen. Our results indicate that the immediate interpretation of FNA biopsies is an accurate procedure that should be routinely employed.     

Reliability of telepathology for frozen section service.

One of the most promising applications of telepathology (pathology at a distance by electronic transmission of images in pathology) is frozen section diagnosis, especially because by means of this tool operations requiring an intraoperative histopathological diagnosis are feasible at hospitals without a pathologist on-site. For the introduction of this diagnostic tool into pathologist's daily practice the evidence of its diagnostic accuracy comparable to that of the conventional frozen section diagnosis is crucial. For this purpose the literature on the diagnostic accuracy of telepathological frozen section diagnosis was reviewed. In a metaanalysis these studies and reports, in which a total of more than 1290 cases had been examined, showed a slightly lower overall diagnostic accuracy (of the telepathological frozen section diagnosis) of about 0.91 than the conventional frozen section diagnosis with an average accuracy of about 0.98 found in an analysis of several studies (on frozen section diagnosis of different organs). This difference is at least predominantly caused by a higher rate of deferred and false negative frozen section diagnoses in the telepathological method, while the specificity of both methods, each more than 0.99 was not significantly different. In conclusion, the introduction of a telepathological frozen section diagnosis for hospitals without an acceptable access to a pathologist is justifiable already at the current state of the technological development especially when considering the advantages (time saving, reduction in costs) compared to the alternative of surgical interventions without access to an intraoperative diagnosis.     

The big problem of the missing cytology slides

Cytology slides are often unique and irreplaceable. Unlike surgical pathology cases, where additional paraffin sections can be cut, cytology slides often cannot be duplicated because there are only a few direct smears or the diagnostic material is present on a single slide. Cytology slides are often "sent out" to other physicians, laboratories or hospitals, typically so that the pathologist at the institution where the patient will receive treatment can review the slides. Less often, a cytology lab sends out the slides for a second opinion or as part of the discovery process in a lawsuit, where they may or may not be defendants. Rarely, unique and irreplaceable cytology slides are lost. This article presents a hypothetical scenario that is based on reported state appellate court decisions. The article discusses some of the legal issues that will affect the defendant cytologist/cytology lab and the "expert cytologist," and suggests some steps a cytologist/cytology lab can take to minimize the risk of repercussions from a lost unique and irreplaceable cytology slide.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

Risk of significant gynaecological pathology in women with ?glandular neoplasia on cervical cytology

A. Talaat, D. Brinkmann, J. Dhundee, Y. Hana, J. Bevan, R. Irvine, S. Bailey and R. Woolas
Risk of significant gynaecological pathology in women with ?glandular neoplasia on cervical cytology Objective: To review the risk of pre‐invasive and invasive gynaecological pathology in women referred with cervical cytology reporting ?glandular neoplasia. Methods: Review of the case notes of all women referred with cervical cytology reported as ?glandular neoplasia between January 1999 and December 2008 at two UK hospitals: Portsmouth Hospitals NHS Trust and Queen Mary’s Hospital Sidcup. The category of ‘borderline nuclear change in endocervical cells’, result code 8 according to the national health service cancer screening programme (NHSCSP), was excluded from the study. Results: A total of 200 women were identified using the hospitals’ pathology computer systems. Invasive carcinoma was found in 48 women (24%): 28 endocervical adenocarcinomas, eight squamous cell carcinomas (SCC), ten endometrial and two ovarian adenocarcinomas. Pre‐invasive neoplasia was found in 115 (57.5%), including 14 cervical glandular intraepithelial neoplasia (CGIN), 31 cervical intraepithelial neoplasia (CIN) grade 2/3 and 70 concomitant CGIN and CIN2/3. CIN1/HPV was found in 25, simple endometrial hyperplasia in three and no histological abnormality in three. Thirty‐four (70.8%) of 48 invasive carcinomas (of which 23 were endocervical adenocarcinomas) were in asymptomatic women investigated for abnormal cytology. Fourteen of 34 (41.4%) of those with ?glandular neoplasia thought to be endometrial were CGIN or CIN2/3. Colposcopic appearances were normal in 47.6% of women with pure cervical glandular neoplasia (adenocarcinoma or CGIN) compared with 12.8% with squamous cell lesions (CIN2/3 or SCC): P = 0.0001. Thus, colposcopy was more sensitive for detecting squamous cell abnormalities than their glandular counterparts. Although cervical adenocarcinomas are less amenable to prevention by screening than cervical SCC, in our study cervical cytology predominantly detected these abnormalities at their early asymptomatic stages. Conclusion: At least CIN2 was found in 81.5% in women referred with cervical cytology reporting ?glandular neoplasia. A thorough evaluation of the whole genital tract is needed if colposcopy is negative.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

Summary The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96%+1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin, Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde+ 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections as regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

Crush cytology of Rosai-Dorfman disease of the central nervous system. A report of 2 cases

BACKGROUND: Rosai-Dorfman disease (RDD) of the central nervous system (CNS) is rare. There is no previous report on intraoperative crush cytology of this disorder. CASES: A 70-year-old man presented with headache and was found to have 2 intracranial extraaxial masses. A 62-year-old man presented with cauda equina syndrome secondary to a mass lesion in the sacral canal. The crush smears, prepared intraoperatively from a small portion of the biopsy specimen in each case, showed scattered lymphoid aggregates in a lighter background with a loose admixture of lymphocytes, plasma cells, usual histiocytes and RDD histiocytes. The RDD histiocytes contained single but sometimes multiple nuclei that were larger and more hyperchromatic than those of the usual histiocytes. Many RDD histiocytes had voluminous, pale pink cytoplasm. Lymphophagocytosis by RDD histiocytes was observed. The findings in the crush smears reflected the histology of the lesions. The lymphophagocytosis and nuclear details in the RDD histiocytes were more visible in the crush smears than in the frozen sections. CONCLUSION: Crush cytology appears useful, alone or in conjunction with frozen sections, in the intraoperative diagnosis of CNS RDD.     

Crush cytology of melanocytoma of the spinal cord. A case report

BACKGROUND: Primary melanocytic neoplasms of the central nervous system are rare. There is no previous report on the intraoperative crush cytology of these neoplasms. CASE: A melanocytoma occurred in a 65-year-old woman who presented with back pain, and weakness and numbness of the right lower extremity. Magnetic resonance imaging revealed an enhancing mass in the conus medullaris. The tumor was subtotally removed. Crush smears revealed dispersed and perivascular aggregates of monotonous oval or spindle tumor cells with round or oval, uniform nuclei; small nucleoli; and long cytoplasmic processes. Some tumor cells contained fine or coarse cytoplasmic brown pigments. The pigments were easier to discern in the crush smears than in the frozen sections. CONCLUSION: Crush cytology appears to be helpful, alone or in conjunction with frozen sections, in the intraoperative diagnosis of meningeal melanocytoma.     

Detection of bladder carcinoma in females by flow cytometry and cytology

The sensitivity of bladder wash flow cytometry (BWFCM), voided urinary cytology (VUC), and cytology of catheterized urine obtained at the time of cystoscopy (CUC) were reviewed on all women evaluated for bladder cancer at Memorial Sloan-Kettering Cancer Center between June 1985 and December 1986. This comprised sixty-four episodes of pathologically proven bladder cancer in 48 women. Considering positive and suspicious results jointly the sensitivities of BWFCM, CUC and 3 VUC were 75%, 64% and 56%, respectively. If only positive results were considered (i.e., suspicious results considered as negative), the sensitivities of BWFCM, CUC and 3 VUC were 64%, 31% and 32%, respectively. The sensitivities of these tests are less than for a predominantly male population, presumably related to the presence of squamous epithelium and greater frequency of pyuria. However, bladder wash flow cytometry and conventional cytology are still a very valuable addition to cystoscopic examination, and the combination of BWFCM with conventional cytology is more sensitive than either procedure alone.     

冰冻切片技术在海滨锦葵细胞学研究中的应用

为解决普通石蜡切片观察植物样品繁琐与耗时长的问题,利用改进的蔗糖保护—液氮速冻切片法,进行海滨锦葵不同器官细胞学研究。结果表明:(1)适合于海滨锦葵不同器官的最适蔗糖浓度不同,适于含水量较大的营养器官(根、茎和叶)的最适蔗糖浓度比含水量较小的花器官(柱头裂片、花柱、子房和花药)高。(2)利用最适蔗糖浓度的蔗糖保护-液氮速冻切片方法,获得了完整而清晰的海滨锦葵各器官组织细胞结构的切片;海滨锦葵具有典型的双子叶草本植物营养器官的组织细胞结构特征,花柱为闭合型花柱,子房为多室复子房。表明基于蔗糖保护-液氮速冻的冰冻切片技术在植物细胞学研究中具有广阔的应用前景。     

Cytopathology of the central nervous system. Part I. Utility of crush smear cytology in intraoperative diagnosis of central nervous system lesions

OBJECTIVE: To evaluate the utility of rapid intraoperative crush smear cytologic diagnosis of central and peripheral nervous system lesions and to determine the accuracy and relevance of the accuracy of the intraoperative cytologic diagnosis when compared to the final paraffin section diagnosis. STUDY DESIGN: The crush (squash) smear technique was introduced at Sher-i-Kashmir Institute of Medical Sciences in May 2003. The 8 months of 2003 were used for standardization of the procedure. In 2004, 151 patients with open neurosurgical specimens or stereotactic biopsies were diagnosed intraoperatively by crush smears, and the diagnosis was compared with final diagnosis on paraffin sections of the same tissue samples. No supplementation of frozen sections was used. RESULTS: Of 151 cases, 144 were diagnosed accurately intraoperatively by crush smear cytology when compared with the respective paraffin section diagnoses. The diagnostic accuracy attained was 95.36%. Each case was diagnosed within 10 minutes after receipt of sample. Neurosurgical procedure (open or stereotaxy) did not affect diagnostic accuracy. CONCLUSION: In the expert hands of a pathologist with good exposure neurosurgical specimens, crush smear cytology is an accura and reliable procedure for the intraoperative diagnosis central nervous system tumors.     

The birth of mice from testicular spermatozoa retrieved from frozen testicular sections

Male gamete cryopreservation has been widely used for both human reproduction and animal breeding. We investigated whether testicular spermatozoa retrieved from frozen testicular sections (10 or 25 mum thick) could support the full-term development of normal progeny. For this purpose, frozen testicular sections were prepared from two genetic backgrounds (BDF1 or B6 GFP transgenic mice), and the functional ability of testicular spermatozoa after preservation for 1 day, 1 mo, and 3 mo was assessed by intracytoplasmic sperm injection (ICSI). Testicular spermatozoa were successfully retrieved from frozen testicular sections for the use of ICSI, regardless of the preservation period. The ICSI technique revealed that oocytes (BDF1 or B6 background) injected with testicular spermatozoa prepared from frozen testicular sections developed into normal progeny, even though the sections had been cryopreserved for 3 mo at -30 degrees C. Approximately 15% and 5% of the embryos preserved for 3 mo developed to full term if the testicular spermatozoa were prepared from the 25- and 10-mum sections, respectively. These results clearly indicate that male gametes can be viably preserved in frozen testicular sections. The technique described herein will allow the preservation of male gametes in the form of a "book" or "file" by mounting the sections on a paper-thin sheet. Furthermore, this technique may be of value in the clinical treatment of severe male infertility, since testicular spermatozoa can easily be found through examination of testicular cross sections rather than by attempts to identify them in testicular cell suspension.     

A nationwide telepathology consultation and quality control program in China: implementation and result analysis

Background

Telepathology may play an important role in pathology consultation and quality control for cancer diagnosis in China, as the country has the largest population of cancer patients worldwide. In 2011, the Pathology Quality Control Center of China and Ministry of Health developed and implemented a nationwide telepathology consultation and quality control program for cancer diagnosis in China. We here report the results of the two-year implementation and experiences.

Methods

the program built an Internet based telepathology platform to connect participating hospitals and expert consultants. The hardware and software used for the platform were validated in previous validation studies in China. The program had three regional centers consisting of Peking Union Medical College, Huasi Medical College of Sichuan and 2nd affiliated hospital of Zhejiang University. It also had 20 provincial consultation centers based in the provincial referral hospitals. 80 provincial or national pathologists served as expert consultants for the program, providing telepathology consultation for cancer diagnosis for more than 60 participating hospitals.

Results

from 2011 to July 2013, 16,247 pathology cases were submitted to the platform for consultation. Among them, 84% were due to diagnostic difficulty and 16% were due to request by patients. The preliminary diagnosis provided by submitting pathologists were in agreement with expert opinion in 59.8% of cases but was in disagreement with expert opinion in 24.2% of cases. 16.0% of cases were not provided with preliminary diagnosis. The distribution of pathology cases by system or organ were: digestive system, 17.3%; gynecologic system, 16.7%; head and neck, 15.7%; bone and soft tissue, 10.4%; lung and mediastinum, 8.6%; breast, 7.6%; urinary system, 7.5%; hematopathology, 6.4%; skin, 5.2%; neuropathology, 2.5% and cytopathology, 1.3%. Expert consultants also provided assessment of quality of slide preparation and staining, online lectures and guidance for pathology quality control.

Conclusion

our results of two years' implementation indicated that telepathology could solve the problem of uneven distribution of pathology resources and provide a solution for countrywide pathology quality control in China. Telepathology could play an important role in improving pathology diagnosis in China.

     

Cytology and clinical spectrum of sexually transmitted infections in Lebanese women as revealed by Pap smear: a cross-sectional study from 2002-2006

To study the cytology profile of cervical smears and the respective prevalence and incidence of certain cervico-vaginal infections detectable by routine Pap smear cytology in Lebanese women from 2002 to 2006. Pap smear cytology results were compiled from the archives of the Institut National de Pathologie for the period extending from 2002 until 2006. This study covered 118,230 cervical specimens obtained from Lebanese women attending clinics and hospitals in all the five districts of Lebanon; prevalence and incidence rates for infections detectable by routine Pap smear examination were determined. A rise in prevalence of these infections by 2.1 percent (2,555) from 2002 to 2006 was revealed. A doubling of Pap smears showing HPV-associated changes was detected (1.4 percent in 2002 to 2.9 percent in 2006), and a simultaneous almost 7-fold increase of Atypical Squamous Cells of Undetermined Significance (ASCUS) cytology was detected during this period; the rise in ASCUS cytology was age-dependent. Moreover, a 60 percent increase in prevalence of bacterial vaginosis (2.3 percent in 2002 to 3.7 percent in 2006) and a more than 3-fold decline in Trichomonas vaginalis infection (1 percent in 2002 to 0.3 percent in 2006) were also noted in this population during this period. An increase in the prevalence and incidence of cervico-vaginal infections detectable by Pap smear cytology in Lebanese women was revealed from 2002 to 2006. Such changes could point to recent modifications of sexual and health behaviours in the Lebanese community.     

How has extracorporeal shock-wave lithotripsy changed the treatment of urinary stones in Quebec?

OBJECTIVES: To determine the number of people who underwent treatment of urinary stones in Quebec before and after the introduction of extracorporeal shock-wave lithotripsy (ESWL) and to determine how the introduction of ESWL influenced resource utilization. DESIGN: Before-after study; data were obtained from administrative databases and hospital-based cost estimates. SETTING: The 68 acute care hospitals in Quebec in which treatment of urinary stones is undertaken. PATIENTS: Quebec residents admitted to hospital for treatment of urinary stones between the fiscal years 1984 and 1992. OUTCOME MEASURES: Number of people treated for urinary stones per year, total number of procedures per year (including open surgery, percutaneous procedures, retrograde procedures and ESWL), and annual resources (including number of hospital bed-days and direct costs) for treatment of urinary stones used overall and in hospitals with and without ESWL services. RESULTS: Over the study period the number of people treated for urinary stones increased by 59%. As well, the combined frequency of ESWL and surgery (the two main treatment methods) increased by 107%. These increases were largely due to rates of treatment that grew by 52% among women and by 34% among men. The total number of hospital bed-days decreased by 28%, which reflected shorter hospital stays for ESWL. However, despite this decrease, the total direct annual costs were 7% higher in 1992 than in 1984 because of the increased numbers of people treated and procedures performed. In the three hospitals that offered ESWL the number of hospital bed-days and the direct costs of treating urinary stones increased by 49% and $2.5 million respectively. In the 65 other hospitals these figures decreased by 41% and about $2.9 million respectively. CONCLUSIONS: Because of increased intervention rates the total cost of treating urinary stones has risen since the introduction of ESWL. The introduction of ESWL has also been associated with a shift in the use of resources for treating urinary stones to hospitals with a lithotriptor. The reasons for the increased intervention rates are unknown. However, given the possibility of negative health effects and the increased costs, studies to determine whether the increased rates improve health outcomes are warranted.     

Fink-Heimer Silver Impregnation of Degenerating Axons and Terminals in Mounted Cryostat Sections of Fresh and Fixed Brains

The silver impregnation method of Fink and Heimer (Brain Res., 4: 369-74, 1967) has been used on cryostat sections of both fresh frozen and formalin-fixed brain tissue mounted on slide. The fixed brains were soaked in 25% sucrose for 2-3 days before freezing. The slides used for mounting were dipped in a 0.5% aqueous gelatin solution containing 50 mg of chrome-alum per 100 ml, drained and allowed to dry in a vertical position. Sections of fresh tissue were fixed for 16 hr in a 10% formalin solution buffered with phosphates to pH 7.0. Staining was carried out according to the Fink-Heimer procedure II and gave results comparable to those obtained on unmounted frozen sections of formalin-fixed material