Does the use of intraoperative frozen section of bone marrow from the cut end of the mandible help assess the adequacy of bone margins following mandibulectomy for oral cancer?

BACKGROUND: The adequacy of the cut end of the mandible following a segmental mandibulectomy done for oral cancer intraoperatively is at times assessed using a frozen section (FS) of the bone marrow (BM) at the cut ends. The study aimed to assess its utility to guide the intraoperative decision on the adequacy of bony margins. MATERIALS AND METHODS: All patients with oral squamous cell carcinoma (OSCC) who underwent segmental mandibulectomy from January 2012 to December 2021 at our institute and for whom intraoperative FS of BM was utilized were included. We analyzed the sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of this in predicting positive bone margins. RESULTS: A total of 457 patients were included in the study. The majority of the cases were per premium cases (n = 372, 81.4%). The median age of the cohort was 52 years (range: 22-80 years). Most patients had T4 disease (n = 406, 88.8%). On FS, BM was positive in only 18 patients (3.9%) for whom the bone margin was revised. BM biopsy report in the final histopathology was positive in 12 patients (2.2%). The sensitivity, specificity PPV, and NPV were 52.3%, 98.65%, 64.7%, and 97.7% respectively. No factors predicting BM positivity on FS could be identified in this cohort. CONCLUSIONS: The BM FS was positive in only a small percentage of patients, and it helped in reducing the bone margin positivity rate from 3.9% to 2.2% only. Hence the intraoperative BM FS seems to have limited utility as seen from our study.

MRI and frozen section evaluation of mylohyoid muscle in determining surgical approach for T2-T3 floor of the mouth cancer.

PURPOSE: The choice of surgical approach for floor of the mouth (FOM) cancer, particularly for intermediate-stage tumors (cT2-cT3), remains controversial. This study aims to evaluate a method considering mylohyoid muscle (MM) invasion as a determinant for surgical approach selection, utilizing magnetic resonance imaging (MRI) preoperatively and frozen section (FS) analysis intraoperatively. METHODS: This observational retrospective cohort study analyzed patients undergoing surgical resection of cT2 and cT3 FOM squamous cell carcinoma (SCC) between January 2013 and June 2023. MM infiltration assessed by preoperative MRI determined the surgical approach: clear infiltration led to compartmental surgery (CS), while doubtful or absent infiltration led to transoral surgery (TOS). Conversion from TOS to CS occurred intraoperatively based on macroscopic evidence or positive FS. Data collected included demographic, clinical, surgical, and pathological variables. Survival analysis was conducted using Kaplan-Meier method. RESULTS: Among 44 patients included, majority had cT2 tumors (59.1%). MM resection was necessary in 22.7% of cases. Overall survival (OS) and progression-free survival (PFS) did not significantly differ between TOS and CS groups. Radiological depth of invasion (rDOI) < 10 mm is correlated with MM preservation in 89% of cases, while rDOI > 10 mm is correlated with MM resection only in 23.8% of cases. Pathological depth of invasion (pDOI) discrepancies were observed in the two groups: in CS group is shown a higher pDOI (> 10 mm) confirmation (90%). Surgical complications and functional outcomes differed between TOS and CS groups. CONCLUSION: Considering MM invasion for surgical approach selection in cT2-cT3 FOM tumors appears oncologically safe, with better functional outcomes in muscle preservation. Preoperative MRI for MM assessment combined with intraoperative FS analysis provides reliable guidance for surgical decision-making.

Challenging pitfalls in frozen section pathology: a case of mandible ghost cell odontogenic carcinoma and the literature review.

BACKGROUND: Ghost cell odontogenic carcinoma (GCOC) is a rare malignancy characterized by the presence of ghost cells, preferably in the maxilla. Only slightly more than 50 case reports of GCOC have been documented to date. Due to the rarity of this tumor and its nonspecific clinical criteria, there is a heightened risk of misdiagnosis in clinical examination, imaging findings, and pathology interpretation. CASE PRESENTATION: A 50-year-old male patient presented to the hospital due to experiencing pain in his lower front teeth while eating for the past 2 months. Upon examination, a red, hard, painless mass was found in his left lower jaw, measuring approximately 4.0 cm × 3.5 cm. Based on the malignant histological morphology of the tumor and the abundant red-stained keratinized material, the preoperative frozen section pathology misdiagnosed it as squamous cell carcinoma (SCC). The surgical resection specimen pathology via paraffin section revealed that the tumor was characterized by round-like epithelial islands within the fibrous interstitium, accompanied by a large number of ghost cells and some dysplastic dentin with infiltrative growth. The malignant components displayed marked heterogeneity and mitotic activity. Additionally, a calcified cystic tumor component of odontogenic origin was observed. Hemorrhage, necrosis, and calcifications were present, with a foreign body reaction around ghost cells. Immunoreactivity for ß-catenin showed strong nuclear positivity in tumor cells, while immunostaining was completely negative for p53. The Ki67 proliferation index was approximately 30-40%. The tumor cells exhibited diffuse CK5/6, p63, and p40 immunoreactivity, with varying immunopositivity for EMA. Furthermore, no BRAFV600E mutation was identified by ARMS-PCR. The final pathology confirmed that the tumor was a mandible GCOC. CONCLUSION: We have reported and summarized for the first time the specific manifestations of GCOC in frozen section pathology and possible pitfalls in misdiagnosis. We also reviewed and summarized the etiology, pathological features, molecular characteristics, differential diagnosis, imaging features, and current main treatment options for GCOC. Due to its rarity, the diagnosis and treatment of this disease still face certain challenges. A correct understanding of the pathological morphology of GCOC, distinguishing the ghost cells and the secondary stromal reaction around them, is crucial for reducing misdiagnosis rates.

DIUTHAME enables matrix-free mass spectrometry imaging of frozen tissue sections.

RATIONALE: A recently developed matrix-free laser desorption/ionization method, DIUTHAME (desorption ionization using through-hole alumina membrane), was examined for the feasibility of mass spectrometry imaging (MSI) applied to frozen tissue sections. The permeation behavior of DIUTHAME is potentially useful for MSI as positional information may not be distorted during the extraction of analytes from a sample. METHODS: The through-hole porous alumina membranes used in the DIUTHAME chips were fabricated by wet anodization, were 5 µm thick, and had the desired values of 200 nm through-hole diameter and 50% open aperture ratio. Mouse brain frozen tissue sections on indium tin oxide (ITO)-coated slides were covered using the DIUTHAME chips and were subjected to MSI experiments in commercial time-of-flight mass spectrometers equipped with solid-state UV lasers after thawing and drying without matrix application. RESULT: Mass spectra and mass images were successfully obtained from the frozen tissue sections using DIUTHAME as the ionization method. The mass spectra contained rich peaks in the phospholipid mass range free from the chemical background owing to there being no matrix-derived peaks in that range. DIUTHAME-MSI delivered high-quality mass images that reflected the anatomy of the brain tissue. CONCLUSIONS: Analytes can be extracted from frozen tissue by capillary action of the through-holes in DIUTHAME and moisture contained in the tissue without distorting positional information of the analytes. The sample preparation for frozen tissue sections in DIUTHAME-MSI is simple, requiring no specialized skills or dedicated apparatus for matrix application. DIUTHAME can facilitate MSI at a low mass, as there is no interference from matrix-derived peaks, and should provide high-quality, reproducible mass images more easily than MALDI-MSI.

What Is Important for Confirming Negative Margins When Resecting Mandibular Ameloblastomas?

PURPOSE: The purpose of this study was to assess the utility of intraoperative radiographs and frozen sections in achieving negative margins and preventing recurrence of mandibular ameloblastomas. MATERIALS AND METHODS: This was a retrospective cohort study of patients who underwent resection (≥1 cm) of mandibular ameloblastomas from 2005 through 2015. Patients were included if they had at least 1-year follow-up and complete records. Demographic variables included age, gender, and type of resection (segmental vs marginal). Predictor variables were type of margin assessment: 1) frozen section, 2) intraoperative ex vivo specimen radiograph, 3) both, or 4) none. The outcome variables were final margin status and recurrence rate. Accuracy of intraoperative radiographic margins was determined by comparison with histologic margin distance. Descriptive statistics were conducted with the Fisher exact test. RESULTS: The study sample consisted of 35 patients (47.5 ± 20.4 yr old; 16 men) who underwent 25 segmental and 10 marginal resections. Ten had frozen sections only, 3 had ex vivo specimen radiographs only, 10 had no intraoperative measurements, and 12 had both. There were no positive frozen sections. One patient had a positive posterior bony margin at final pathology despite negative frozen section histology. There was no difference in recurrence rate at latest follow-up among cohorts. The anterior radiographic margin was 11.8 ± 5.9 mm compared with 11.5 ± 7.5 mm by histology (P = .124). The posterior radiographic margin was 12.3 ± 5.3 mm compared with 9.8 ± 6.5 mm histologically (P = .546). Margin distances that were at least 5 mm when measured with specimen radiographs had histologic margin distances of at least 5 mm in 25 of 30 resection margins (83.3%). CONCLUSION: Resection of ameloblastoma with planned margins of at least 1 cm is sufficient to prevent recurrence of ameloblastoma. Achieving a radiographic margin of at least 5 mm provided a histologic margin of at least 5 mm 83.3% of the time.

[Benefits and the accuracy of the intra-operative frozen section at suspected ovarian tumours]. / Prínos a validita peroperacní frozen section u suspektních ovariálních tumoru.

OBJECTIVE: To assess benefits and the accuracy of the intra-operative frozen section for the operative strategy at suspected ovarian tumours. TYPE OF STUDY: Retrospective study. SETTING: Department of Obstetrics and Gynaecology, Faculty of Medicine and Dentistry, Palacky University in Olomouc. MATERIALS AND METHODS: Intraoperative frozen section and final histopathology were compared in 53 patients who underwent operative treatment for suspected ovarian neoplazma. The accuracy of the frozen section findings was evaluated according to the histopatological type of tumours and categories - benign, malignant and borderline tumours. CONCLUSION: In accordance with literature reports a good reliability of the frozen section as for the sensitivity, specificity, positive and negative predictive values were proved. The majority of errors occured in diagnosing mucinous and borderline tumours.

Glial network responses to polymicrobial invasion of dentin.

This study investigated the distribution patterns of glial networks disclosed by reactivity for glial fibrillary acidic protein (GFAP) and S100B in healthy and carious human teeth. The objective was to determine the assembly and collapse of glial networks in response to encroaching infection. 15 healthy and 37 carious posterior teeth from adults were studied. Immediately after extraction, teeth were cleaned and vertically split and the half with pulp fixed and prepared for resin or frozen sections. Sections were stained with toluidine blue and for immunofluorescence, with observation by confocal laser microscopy and analysis by ImageJ software. Carious teeth were subdivided into three groups according to degree of carious involvement: microbial penetration through enamel (stage A), extension into dentin (stage B) and advanced penetration into dentin but without invasion of underlying pulp tissue (stage C). In stage A lesions there was marked increase in glial networks in dental pulp tissue that extended beyond the zone of microbial invasion. This response was maintained in stage B lesions. In advanced stage C lesions these networks were degraded in the zone of invasion in association with failure to contain infection. Cells expressing the glial markers GFAP and S100B showed a response to initial microbial invasion of dentin by increase in number and altered anatomical arrangement. The late stage of dentinal caries was marked by collapse of these networks in the region adjacent to advancing bacteria. This behaviour is important for understanding and explaining the defensive response of the neurosensory peripheral dental pulp apparatus to infection.

The sensitivity and specificity of frozen-section histopathology in the management of benign oral and maxillofacial lesions.

PURPOSE: The management of odontogenic cysts and tumors typically requires a biopsy, which may present significant challenges and prompt an additional visit to the operating room before definitive treatment. The aim of this study was to determine the validity of frozen-section diagnosis in the management of benign oral and maxillofacial lesions, allowing intraoperative diagnosis followed by definitive treatment under the same general anesthetic. MATERIALS AND METHODS: A retrospective chart review of patients treated at the University of Michigan Health System was performed. Patients of all ages who had a diagnosis of a benign maxillofacial lesion by frozen-section and permanent histopathology reports were included for analysis. Patients were identified using the Current Procedural Terminology code for enucleation and curettage and International Classification of Diseases, Ninth Revision codes for benign cysts or tumors of skull, face, or lower jaw. RESULTS: Of 450 patients reviewed, 214 had intraoperative frozen-section examination available for comparison with permanent histopathology. There were 121 men (56.5%) and 93 women (43.5%), with a mean age of 41 years. Compared with final permanent histopathology, the overall sensitivity of frozen sections was 92.1%. Frozen-section histopathology had a sensitivity greater than 90% and a specificity greater than 95% for the diagnosis of dentigerous cyst and keratocyst odontogenic tumor. CONCLUSIONS: In this study of 214 patients with benign maxillofacial lesions, frozen-section histopathology was found to be a valid diagnostic modality with high sensitivity, specificity, and positive and negative predictive values. These results and analysis support the use of frozen-section histopathology for the treatment of benign maxillofacial lesions and underscore its value in the management of these lesions.

Quantitative analysis of the terminal branches of facial nerve in fresh frozen head and neck specimens.

BACKGROUND: The first aim of this study was the quantification of nerve fibres found in terminal branches of facial nerve and the second aim was the ultrastructural analysis of these terminal branches in order to observe their ultrastructural differences, if present. In the examination of literature; we could not find any studies related to this subject. MATERIALS AND METHODS: Four fresh frozen head and neck specimens were used and the dissections were done bilaterally. Therefore; totally 8 samples were examined. The samples were prepared according to routine transmission electron microscopic tissue preparation technique. The semi-thin sections were examined under light microscope by camera lucida. In every sample, the quantitative analysis was performed in 5 different areas in an area of 0.01 mm2 and statistical analysis was done. Secondly; the ultrastructural appearance of these terminal branches were examined under transmission electron microscope. RESULTS: In the quantitative analysis of terminal branches of facial nerve in an area of 0.01 mm2; the least number of nerve fibres were found in temporal branches and the highest number were detected in cervical branches. In transmission electron microscopic examination, no significant difference was found in between these branches. In the statistical analysis; statistically significant differences were obtained in between the temporal and buccal, marginal mandibular, cervical branches; zygomatic and marginal mandibular, cervical branches; buccal and marginal mandibular, cervical branches; marginal mandibular and cervical branches. CONCLUSIONS: These numerical data will have an importance during the nerve repair process of terminal branches of facial nerve in various injuries.

[Evaluation of shrinkage of surgical margins on tongue specimens following formalin fixation]. / Formalin fiksasyonu sonrasi dil örneklerinde cerrahi sinir küçülmesinin degerlendirilmesi.

OBJECTIVES: This study aims to evaluate the alterations in distances to the surgical margins on sheep tongue specimens, through resection, formalin fixation, frozen section, and microscopic examination. MATERIALS AND METHODS: Fifty fresh sheep tongues were used in the study. A metal plate was fixed on the lateral aspect of each tongue to represent tumor tissues. A total of 40 specimens with either 1 cm or 2 cm distances as surgical safety margins of the surrounding plate were prepared using either scalpel or monopolar cautery (10 specimens were prepared for each group). Additional 10 specimens with 1 cm safety margins were prepared using either scalpel or monopolar cautery (5 specimens per group) for frozen section examination. Distances to the metal plates before resection were compared with the ones which were determined after resection, frozen section examination, 24-hour formalin fixation and microscopic examination. RESULTS: Distances to the surgical margins were found to be decreased in all specimens after resection, 10% formalin fixation and microscopic examination. The distances to the surgical margins were observed to be reduced by 6.5-7.5% on average after resection, 10-12% on average after formalin fixation and 30% on average after microscopic examination, compared to the baseline values. The level of shrinkage was reduced by 6.3-10% following microscopic section preparation during frozen section examination. CONCLUSION: Seven to eight-millimeter distance to the surgical margin at minimum should be maintained to achieve a 5 mm in height surgical safety margin during sheep tongue resection. The distance defined by the pathologist may be multiplied with 1.42 to estimate around in-situ distance to the surgical margins. Therefore, 1.42 may be used as a corrective factor for sheep tongue tissues.

Intraoperative evaluation of the resected bone margin in mandibular cancers using a trephine drill and frozen section analysis.

The objective of this study was to evaluate the accuracy and feasibility of intraoperative frozen section analysis of samples harvested with a trephine drill from the bone resection margins to identify malignancy. Thirty-five patients who were diagnosed with locally advanced oral squamous cell carcinoma involving the mandible were included in this study. After tumour resection, bone samples were collected from the resection margin of the specimen using a trephine drill. Sampling yielded a cylindrical specimen of bony tissue that included both cortical and cancellous areas. A second sample was obtained from the area where bone invasion was evident; this was used as a positive control. Frozen section analysis was performed intraoperatively to check for malignancy. The sensitivity of this technique was found to be 81.8%, with specificity of 87.5%, a positive predictive value of 75%, negative predictive value of 91.3%, and accuracy of 85.7% when compared to standard histopathology as the gold standard. In conclusion, the evaluation of bone margins using the trephine drill technique and frozen section analysis proved to be fast and reliable.

Preoperative incisional and intraoperative frozen section biopsy techniques have comparable accuracy in the diagnosis of benign intraosseous jaw pathology.

PURPOSE: To compare the accuracy of intraoperative frozen section (FS) and preoperative incisional biopsy (IB) techniques to diagnose benign intraosseous jaw lesions. MATERIALS AND METHODS: This is a retrospective cohort study composed of subjects with benign intraosseous jaw lesions. The predictor variable was the technique for establishing a preliminary diagnosis of the lesion, preoperative IB or intraoperative FS. The outcome variable was the accuracy of the biopsy technique when compared with the final histologic diagnosis and was classified as concordant or discordant. The comparative diagnostic accuracy of the techniques was assessed with the χ(2) test. RESULTS: A total of 71 subjects met inclusion criteria. The mean age was 39 years (range, 5 to 85 years), and 58% (41) were male patients. Of the subjects, 20 (28%) underwent IB. In 14 (70%) of these, the results of biopsy agreed with the final diagnosis. 51 (72%) underwent intraoperative FS and in 31 (62%) of these, the results of biopsy agreed with the final diagnosis. The difference in diagnostic accuracy between IB (70%) and FS (61%) was statistically insignificant (P = .48). Sources of biopsy error included sampling error (46%), insufficient epithelial tissue (15%), inflammation (15%), pathologist's experience (8%), and artifact (4%). CONCLUSIONS: Preoperative IB and intraoperative FS provide comparable accuracy of diagnosis in patients with benign intraosseous jaw pathology. Sampling error was the most common reason for discordant results.

Effectiveness of fresh frozen and cryopreserved homologue iliac crest grafts used in sinus lifting: a comparative study.

In the last decade, several investigators have reported that autologous and homologous fresh frozen bones (FFB) are effective materials to restore alveolar ridges previous to insert dental implants. Recently we have used cryopreserved homologue grafts (CFFB). Here we reported a retrospective comparative study between implants inserted in FFB and CFFB evaluate their clinical outcome. Patients were treated with a split mouth scheme for bone grafting with FFB and CFFB and spiral family implants (SPI) were inserted in the same surgical time. Several variables (patient, grafts, anatomic site, implant, prosthetic restoration) were investigated. Implant' failure and peri-implant bone resorption were considered as predictor of clinical outcome. A total of 84 SFIs were inserted in 12 patients. Implants were inserted to replace 8 incisors, 4 cuspids, 31 premolars and 41 molars. The mean follow-up was 14 months. Three out of 84 implants was lost (i.e. survival rate SVR = 96.4%) and no differences were detected among the studied variables. Similar result was obtained by analyzing the crestal bone resorption around implant' neck (i.e. success rate). FFB and CFFB have high and comparable survival and success rate. Implants inserted with one step surgical procedure in native (i.e. not grafted) bone, FFB and CFFB have similar clinical outcome.

Pulmonary Aggregatibacter actinomycetemcomitans infection masquerades as malignancy in a patient with periodontitis.

A 49-year-old man with a 37.5 pack-year smoking history presented with a suspected neoplasm of the right lung following the discovery of a metabolically active mass on positron emission tomography-CT imaging. The patient, who demonstrated poor oral hygiene, had a history of irregular problem-oriented dental visitation. Having excluded malignancy through histologic investigations, Aggregatibacter actinomycetemcomitans-a well-established periodontal pathogen-was subsequently cultured from his pulmonary aspirate. The patient was therefore managed with systemic antimicrobials and adjunctive dental extractions to eliminate the likely source of infection, whereafter the mass resolved without complication. This case corroborates previous reports of extraoral isolation of A. actinomycetemcomitans, which may mimic cancer clinically and radiographically. While a definitive causative link between untreated periodontitis and systemic infection remains to be elucidated, such cases present a compelling argument in favour of promoting oral health to prevent systemic disease.

Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis.

BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. MATERIAL AND METHODS: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. RESULTS: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. CONCLUSION: We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.

[Application of polymer detection systems of Histofine MOUSESTAIN, Mouse MAX-PO and rat MAX-PO for mouse and rat frozen tissue sections].

We defined the staining applicability of Histofine MOUSESTAIN, Mouse MAX-PO and Rat MAX-PO for fixed frozen and fresh frozen tissue sections besides paraffin embedded tissue sections. Two different types of cool air dried tissue sections, fresh frozen tissues and fixed frozen tissues of nine-week old Slc: ICR mouse and nine-week old Slc: SD rat, were prepared for immunohistochemistry (IHC) staining in accordance with conventional method. Additional two steps of 1 and 2 to our recommended procedure for paraffin embedded tissue sections were examined as follows: STEP 1: Adjust reaction time of polymer; STEP 2: Add 0.2% of glutaraldehyde (GA) solution before the STEP 1, when background staining is still observed by STEP 1. The steps 1 and 2 resulted in elimination of background staining of most Histofine series. Therefore, polymer detection systems, Histofine series, are applicable for IHC staining of two different types of frozen tissue sections as well as paraffin embedded tissue sections.

A novel protocol to detect green fluorescent protein in unfixed, snap-frozen tissue.

The green fluorescent protein (GFP) is a powerful reporter protein that allows labeling of specific proteins or entire cells. However, as GFP is a small soluble protein, it easily crosses membranes if cell integrity is disrupted, and GFP signal is lost or diffuse if the specimen is not fixed beforehand. While pre-fixation is often feasible for histological analyses, many molecular biology procedures and new imaging techniques, such as imaging mass spectrometry, require unfixed specimens. To be able to use GFP labeling in tissues prepared for such applications, we have tested various protocols to minimize the loss of GFP signal. Here we show that, in cryocut sections of snap-frozen brain tissue from two GFP reporter mouse lines, leaking of the GFP signal is prevented by omitting the commonly performed drying of the cryosections, and by direct post-fixation with 4% paraformaldehyde pre-warmed at 30-37 °C. Although the GFP staining does not reach the same quality as obtained with pre-fixed tissue, GFP localization within the cells that express it is preserved with this method. This protocol can thus be used to identify GFP positive cells on sections originating from unfixed, cryosectioned tissue.

Enzyme-labeled antigen method: histochemical detection of antigen-specific antibody-producing cells in tissue sections of rats immunized with horseradish peroxidase, ovalbumin, or keyhole limpet hemocyanin.

The enzyme-labeled antigen method is a histochemical technique that visualizes antigen-specific antibody-producing cells in tissue sections, originally documented in 1968. In this study, we attempted to reemerge this hidden but potentially useful method in rat models immunized with horseradish peroxidase (HRP), ovalbumin (OA), or keyhole limpet hemocyanin (KLH). After repeated immunization in footpads, popliteal, groin, and axillary lymph nodes and spleen were sampled. Paraformaldehyde-prefixed frozen sections were incubated with HRP, biotinylated OA, or biotinylated KLH. Proteinase K pretreatment and the secondary use of HPR-labeled streptavidin were applied in the latter two situations. Plasma cells producing antigen-specific antibodies were visualized. Proportions of antigen-specific antibody-producing cells in total plasma cells shown with the immunoperoxidase method for rat immunoglobulins were evaluated. The percentage of antigen-specific plasma cells reached approximately 50% of total plasma cells in the regional lymph nodes. The specificity was confirmed by (a) negativity in non-immune rat tissue, (b) negativity with indifferent antigen probes, and (c) abolishment of the reactivity with the corresponding rat serum. In buffered formalin-fixed, paraffin-embedded tissues, fewer plasma cells were labeled for HRP and KLH antibody reactivity after strong proteolysis and prolonged incubation. Expectedly, this method allows us to observe antigen-specific antibody-producing cells under varied pathological conditions.

Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway.

Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.