Does p53 immunostaining improve diagnostic accuracy in urine cytology?

The frequent change of the transitional cell carcinoma of the urinary tract accounts for the fact that cytological abnormalities in urinary specimens are often not sufficient to enable a definitive diagnosis of malignancy. The purpose of this work was to evaluate the possible use of p53 protein in increasing the diagnostic accuracy of urinary cytology. The expression of p53 was investigated by immunocytochemistry in two groups of urinary specimens, one cytologically positive and the other cytologically negative for cancer. Immunostaining was carried out using a monoclonal antibody to p53. In the positive group, in which bladder cancer was confirmed by cystoscopy and biopsy (31 cases), positive reaction for p53 was found in 55% of the cases (17 cases). In the negative group (92 cases), presence of cancer was histologically ascertained in 64 cases and in this group 15 cases (23.4%) showed positive p53 staining. In the remaining 28 cases of this group, where TCC was not present, 7 cases showed p53 positivity in non-neoplastic urothelial cells. This result shows that, while immunocytochemical detection of p53 in urinary specimens may be used for prognostic evaluation of patients with bladder cancer, it does not contribute to the diagnostic accuracy in cases with morphologically inconclusive or negative cytology. The sensitivity and specificity of the method in detecting bladder carcinoma were 23.5 and 75%, respectively.     

Stromal fragments in invasive carcinoma. Source of diagnostic difficulty in aspiration cytology

OBJECTIVE: To analyze three cases of stromal fragments in invasive carcinoma that created diagnostic difficulty in aspiration cytology. STUDY DESIGN: A retrospective review of fine needle aspiration cytology (FNAC) smears of a breast tumor, scalp tumor and neck mass. Cytomorphologic features of all the smears were reviewed after histology became available. RESULTS: FNAC smears revealed a biphasic pattern: a carcinomatous component and a stromal component that was either discrete or in close apposition to the carcinoma. The cytopathologist had suggested the diagnosis of a biphasic tumor in each case--phyllodes, malignant skin adnexal and salivary gland tumor. Histopathology revealed an invasive carcinoma with altered stroma in the first two cases and metastatic lymph node with perinodal soft tissue extension in the third case. CONCLUSION: Stromal changes in response to infiltrating carcinoma are well documented in surgical pathology. However, these may also be encountered in FNAC smears. The above cases stress the importance of recognizing stromal fragments in aspiration cytology in order to avoid diagnostic errors.     

Testicular fine needle aspiration cytology as a diagnostic tool in dog infertility

The results of testicular aspirate cytology taken from clinical patients with a history of infertility were compared with the clinical and histological findings. Azoospermia was the most common and the most rewarding indication for the examination. Samples were also taken from cases with suspected testicular tumours, orchitis, epididymitis, severe oligo- and teratozoospermia, lack of libido and unilateral testicular atrophy. Histological and cytological findings were found to correlate well. Identification of cell types from normal germinal epithelium was relatively easy. No immediate adverse effects of aspiration were noted. Five normospermic dogs were monitored for two to six months after aspiration, and there were no marked deleterious effects on testicular consistency, testicular histology or semen characteristics. Testicular cytology obtained by fine needle aspiration may, at least to some extent, be used to assist clinical diagnosis, especially in azoospermic dogs and dogs with palpable changes of testicular tissue.     

A UK-wide trial of the Banff classification of renal transplant pathology in routine diagnostic practice

BACKGROUND: The Banff classification of renal transplant pathology has gained wide support since its introduction in 1993. There have been several studies which have tested its usefulness in the context of research-oriented centres. We sought to evaluate its use in a wider context. METHODS: We recruited pathologists from all but one of the renal transplant centres in the UK. Sections were circulated from 21 selected, 'difficult' cases, in all of which the clinical question was confirmation or exclusion of acute rejection, and in all of which a definite diagnosis had been obvious from the subsequent clinical course. Participants were asked first to diagnose or exclude acute rejection by their usual approach, then to apply the Banff classification. No clinical information was given beyond the time since engraftment, in order to confine the evaluation to the morphological features present in the sections. At the end of the study the subjective impressions of the participants were sought using a structured questionnaire. RESULTS: Using the Banff classification produced no detectable difference in the number of 'correct' diagnoses when compared with a conventional approach, irrespective of whether the 'correct' diagnosis is based on retrospective clinical information or on the consensus opinion of the pathologists involved, and irrespective of where in the Banff schema one applies a 'cut-off' for the diagnosis of acute rejection. However, the reproducibility of the diagnoses was improved. The results suggest that in the Banff classification the best 'cut-off' for the diagnosis of acute rejection is between Banff category 3 and category 4, although in this difficult area we found a large improvement in diagnostic accuracy if input of clinical information occurs. CONCLUSIONS: The improved reproducibility justifies the use of the Banff classification to harmonise approaches between centres, especially in research projects. While there are good reasons also to adopt it in routine diagnostic practice, further refinement is necessary before an improvement in the accuracy of diagnosis can be demonstrated.     

The role of immunocytochemistry in congenital myopathies

The use of stents does not appreciably improve restenosis (usually resulting from intimal hyperplasia) as compared to percutaneous transluminal angioplasty (PTA) alone. The development of small-caliber probes for afterloading therapy in the biliary tract allowed us to use these for therapy in the vascular system. Using a special 9 F catheter, exact measurement of the length of the stented vascular segment and of the insertion length of the afterloading probe could be reproducibly performed. We used a Nucletron (Micro) Selectron HDR planning system version 10.10 for exact calculation, monitoring, and control of the afterloading procedure. Our source was iridium 192 (10 Ci) with a diameter of 1.1 mm. The program controls and monitors the insertion and removal of the iridium probe from the source into the special catheter through to the tip, and monitors the irradiation duration. The exposure time was around 200 seconds for a surface dose of 12 Gy. To date, a total of 40 patients have been treated with endovascular afterloading. All patients suffered from clinically relevant reocclusions or restenoses in stented vascular segments of the superficial femoral artery following successful PTA or laser treatment, within 6 to 8 months after the last therapy. In all patients it was possible to perform re-PTA treatment without remaining residual stenoses in the stented region. The additional time required as compared to PTA alone was approximately 45 minutes with most of this time spending for transportation between the cath lab and afterloading room. The follow-up period of the 40 patients ranged from 4 months to 71/2 years. In 33 patients, there was no deterioration of the clinical stage and no restenosis. One patient suffered from an acute thrombosis approximately 3 months after stent implantation, another patient had a stenosis 3 cm above the stented vascular segment 12 months after irradiation treatment. Follow-up examinations have revealed no evidence of nerve lesions following irradiation therapy. The tissue surrounding the artery showed no change following irradiation therapy, either in the CT, color-coded Doppler, endovascular ultrasonic scan or MRI. No complaints of discomfort were reported during or after irradiation. With the exceptions mentioned above, there was no evidence of any complications.     

Angiosarcoma at unusual sites. A report of two cases with aspiration cytology and diagnostic pitfalls

BACKGROUND: Angiosarcomas are uncommon soft tissue neoplasms with a predilection for skin and superficial soft tissues. CASES: Two cases of angiosarcoma occurred at unusual sites, the parotid gland and lung. The parotid lesion was characterized by malignant cells present singly, in loose groups, in tight three-dimensional aggregates and in acinar formation initially misinterpreted as an adenocarcinoma. The lung mass showed malignant cells in association with vascular endothelium, suggestive of angiosarcoma. Both cases were negative for Ulex europaeus and Factor VIII-related protein but demonstrated strong immunopositivity for CD31, a highly specific endothelial marker. CONCLUSION: In the absence of vasoformative structures, important diagnostic pitfalls are pseudovascular adenoid squamous cell carcinoma, poorly differentiated adenocarcinoma, melanoma and lymphoma. Immunocytochemical studies and clinical history are essential to the correct diagnosis.     

Multiparameter flow cytometric DNA analysis of effusions: a prospective study of 36 cases compared with routine cytology and immunohistochemistry

Single-parameter flow cytometry (SFCM) is limited in its ability to detect aneuploid and diploid malignant cells or accurately estimate S-phase fractions (SPF) in effusions because of the high degree of contamination by benign mesothelial cells and inflammatory cells. We examined 36 pleural and peritoneal fluids by conventional cytology and multiparameter FCM (MFCM) to analyze the DNA content of cells expressing epithelial markers cytokeratin, epithelial membrane antigen, carcinoembryonic antigen, BRST-1, or BRST-3 (B72.3) and compared the results to those found with SCFM. The cases were also studied by immunohistochemistry using the same antibody panel. By routine cytology, 14 of the 36 cases were classified as carcinomas, 11 as reactive, 1 as mesothelioma, and 10 as suspicious. MFCM allowed reclassification of 5 of the 10 suspicious cases as carcinomas and the remaining 5 as reactive cases based on ploidy and marker expression. Whereas SFCM detected only 13 nondiploid carcinomas, MFCM detected 4 diploid and 15 nondiploid carcinomas. All reactive cases were diploid by SFCM or MFCM. The mesothelioma case showed were distinct peaks by MFCM, a diploid peak with SPF of 13.4% and a tetraploid peak with SPF of 36.1%. The SPF of the nondiploid carcinomas ranged from 5.9 to 50.4% and diploid carcinomas, from 3.5 to 14.5% when gated on epithelial cells. The reactive cases had SPF ranging from 0.4 to 4.4%.(ABSTRACT TRUNCATED AT 250 WORDS)     

The AutoPap system for primary screening in cervical cytology. Comparing the results of a prospective, intended-use study with routine manual practice

OBJECTIVE: To evaluate the effectiveness of the AutoPap System in detecting abnormal and normal cervical smears when used in a primary screening/quality control mode, as compared with currently established laboratory practices. STUDY DESIGN: Slides were obtained prospectively and were initially processed in the routine fashion with cytotechnologist screening followed by 10% random quality control rescreening. Slides were then processed on the AutoPap System and allocated into the following groups: (1) approximately 25% of the lowest-ranking slides were placed in the laboratory's archives as within normal limits; (2) the remaining approximately 75% of slides were subjected to manual screening. Approximately 15% of the highest-ranking slides in this group underwent quality control rescreening. For each slide needing manual screening, the cytotechnologist was supplied with a report giving the ranking score of that slide. All discrepant slides for either adequacy or diagnosis were subjected to a truth-determination process. The results obtained from the two arms of the protocol were then compared. RESULTS: The AutoPap System-assisted arm of the study was superior to the current practice arm for the identification of abnormal slides at the level of atypical squamous cells of undetermined significance and above (ASCUS+), low grade squamous intraepithelial lesion (LSIL) and higher LSIL+. AutoPap System-assisted practice was equivalent to current practice for the identification of unsatisfactory and satisfactory but limited by slides. All results showed statistical significance. In addition, AutoPap System-assisted practice in the study indicated improved specificity of diagnosis. CONCLUSION: AutoPap System-assisted practice shows superior sensitivity and specificity when compared to current practice. Its clinical use as a primary screening device should improve the overall practice of cervical cytology as well as provide potential enhancement in overall laboratory productivity.     

Detection of infectious disease agents in tissue by immunocytochemistry

1. Immunocytochemical procedures have played an increasingly larger role in the identification of infectious disease agents in tissue sections owing to the increased availability and specificity of antibody reagents, the great sensitivity of the methods, and the relative facility with which the studies are performed. 2. Immunocytochemical methods can be applied to routine formalin-fixed tissue for the detection of infectious agents such as viruses, bacteria, fungi, and protozoa among other microorganisms for diagnostic and research purposes.     

Assessment of testicular cytology by fine-needle aspiration and the imprint technique: are they reliable diagnostic modalities?

OBJECTIVE: To investigate whether testicular cytology may be considered diagnostic in the evaluation of infertile men. PATIENTS AND METHODS: Specimens of testicular tissue obtained either surgically (imprint smear) or through fine-needle aspiration (FNA) were used as a source of cytological smears; 58 testes from 24 men with azoospermia or severe oligospermia and from five men with advanced prostate cancer were evaluated cytologically and compared with the histological diagnosis. RESULTS: FNA caused no apparent trauma. The results from FNA smears generally agreed with the histological findings but four patients with no spermatozoa in the FNA smears were diagnosed histologically as hypospermatogenic and two others judged histologically as having Sertoli-cell-only (SCO) syndrome and spermatogenic arrest had detectable spermatozoa in their FNA smears. There was complete agreement between the results of imprint smears and histological findings in those patients with SCO syndrome and spermatogenic arrest. There were no evident differences in sperm counts between hypospermatogenesis and normal spermatogenesis on the imprint slides, but FNA smears detected this difference. CONCLUSION: FNA of the testis is a relatively non-invasive and reproducible technique for evaluating qualitative and quantitative cytology. However, it is insufficient for diagnosing some testicular pathologies. Imprint smears supplement the histological diagnosis, especially if the histological slides are stained unsatisfactorily.     

The increasing power of immunohistochemistry and immunocytochemistry

Since its introduction in the early 1940s, immunostaining technology has developed in a remarkable way, and the applicability of immunohisto/cytochemical probing methods will unquestionably continue to increase in several directions. Immunofluorescence remains the most powerful and reliable immunohistochemical approach for multicolour staining to evaluate co-localization of two or more antigens in an objective manner. Moreover, the fluorescent colour signals exhibit a relatively consistent relationship to the actual antigen concentration in the test preparation and are hence better suited for quantitative computerized image analysis than light-microscopic observations of immunoenzyme staining. On the other hand, immunoenzyme methods are more economical with regard to reagent consumption and are therefore ideal for the use in semiautomatic staining machines. In addition, the superior morphological correlate provided by the latter methods, makes them more attractive and adequate for most purposes in diagnostic pathology laboratories. However, multicolour immunoenzyme staining provides an easily obtainable and reliable result only when the antigens are known a priori to be separately located, both because of technical problems and because imbalanced colour mixing is difficult to evaluate in the light microscope. All these aspects of immunohistochemistry are briefly reviewed in this historical perspective coloured by the author's own experience.     

Sperm cytology in azoospermia

BACKGROUND: To progress the clinical treatment of neonates, especially in the management of respiration, we have to be able to measure their pulmonary function appropriately. Various methods have been developed, but little is known about the pulmonary function of very low birthweight infants (VLBWI) because of the difficulty in taking their measurements with existing equipment. We have developed a very low dead space pneumotachograph to measure lung function in VLBWI. METHODS AND RESULTS: We used our pneumotachograph on 30 infants each weighing less than 1500 g at birth. The infants were intubated with endotracheal tubes of 2.5 or 2.0 mm diameter to measure tidal volume and minute ventilation in the prone and supine position. The tidal volume in the supine position was 6.99 +/- 0.42 mL/kg and 7.58 +/- 0.38 mL/kg in the prone position (mean +/- SE). The tidal volume was significantly larger in the prone than the supine position (P < 0.05). However, no significant difference was observed in minute ventilation and respiratory rates. CONCLUSION: The tidal volume significantly increased in the prone position in VBLWI, confirming the previous observation of larger healthy infants is also applicable to the very low birthweight infants.     

Cervical cytology in adolescence

To further define the chemical structure of human endogenous digoxinlike immunoreactive factors (DLIF) we used human pleural effusions as a source of the substance. Digoxinlike immunoreactive factor activity was detected by radioimmunoassay in the pleural fluid of each of four patients; average concentration was 0.35 ng/mL. The chemical profile of DLIF was determined by initial extraction and concentration of DLIF by ion exchange chromatography followed by reverse phase-high-pressure liquid chromatography (RP-HPLC) separation and purification. Using high-pressure liquid chromatography cochromatography of DLIF, together with several radioactively marked glycosides, we observed a single peak of DLIF activity that was chromatographically identical to digoxin. The present study further supports the recent finding that DLIF is related structurally to the cardiac glycosides, and for the first time it has been proven that DLIF is present in pleural fluids.     

Malignant melanoma in the CNS, subtyping and immunocytochemistry

Paraffin-embedded specimens from 21 patients (mean age 49 years) with malignant melanocytic tumors of the central nervous system were studied. Extraneuronal primary tumors were situated at the trunk (38%), the lower (14%) or upper extremity (10%), and the head/neck region (5%). In 33% no extraneural primary tumor could be detected. The tumor location was frontal (19%), occipital (19%), parietal, spinal, multifocally (14%, respectively), or temporal (5%). Four subtypes were distinguished according to the predominant histological cell type: pleomorphic, epithelioid, spindle- and mixed-cell tumors. 29% contained no melanin, most of them belonging to the epithelioid subtype. The morphology and immunohistochemical reactivity for different antibodies (KL-1, EMA, VIM, HMB-45, NKI-C3, S-100, and MIB-1/Ki-67) were assessed. Positive staining was demonstrated for HMB-45 (in 86% of cases), NKI-C3 (100%), S-100 (95%), vimentin (75%), and KL-1 (33%). No expression of the cytokeratin EMA could be detected. The mean proliferation index measured by MIB-1 immunoreactivity was 21%. The 4 histological subtypes were found to express different antigen patterns. In the analysis of CNS tumors of unknown origin, the panel of antibodies used for diagnosis should include HMB-45 as the most specific marker for malignant melanoma.     

Freeze substitution after fast-freeze fixation in preparation for immunocytochemistry

As compared to classical chemical fixation, the physical immobilization of ultrastructures by fast-freeze fixation (FFF) and the subsequent exchange of water in its solid state by freeze substitution (FS) improve the preparation procedure for immunogold labeling (IGL). FFF-FS results in a morphological preservation of unchallenged quality, as well as in a better preservation of antigenic reactivity, thus allowing remarkable precision of labeling on sections. However, FFF, particularly over a cooled metal plate, requires a heavy and expensive machine. It is not suitable for all biological specimens and in the best conditions, which remain difficult to standardize, the thickness of the well-preserved portion of the specimen does not exceed a few microns for compact tissues, and exceptionally 30-40 microns for isolated cells. The FS procedure is long and must be adjusted empirically for every new specimen and antigenic detection. The preservation of a given antigen's reactivity in the presence of fixative agents and embedding resins remains unpredictable. The action of fixative agents is different and milder in FS than when they are used classically in chemical fixation. By chance, one of the best FS procedures for the preservation of both ultrastructure and antigenicity appears to be by using acetone alone, together with a molecular sieve to improve the water exchange process. A large choice of embedding resins usually allows us to find a compromise between ultrastructural and antigenic preservation.     

Impression cytology in atopic dermatitis

PURPOSE: This study aimed to describe the ocular surface disorder in patients with atopic dermatitis (AD). DESIGN: A prospective case-controlled study. PARTICIPANTS: A total of 44 patients with active AD seen at Kobe University School of Medicine, Department of Ophthalmology, during 1994 through 1996 and 22 normal control subjects were studied. INTERVENTION: The subjects underwent routine ophthalmic examinations, tear film break-up time (BUT), Schirmer test, and conjunctival impression cytology. MAIN OUTCOME MEASURES: Patients and control subjects were compared for tear function parameters, goblet cell density, and conjunctival squamous metaplasia grade. The relation of duration and recurrences of AD to the ocular surface disorder also was looked for. RESULTS: The duration of atopic disease ranged from 18 to 32 years (mean, 22.8 years). The average for exacerbations was 4.5 times. Chronic allergic conjunctivitis with superficial punctate keratitis was the most frequent clinical presentation. The BUT and Schirmer test values were significantly lower in patients with AD compared with those of the control subjects. Impression cytology showed goblet cell loss and conjunctival squamous metaplasia, both of which related to the number of recurrences of AD rather than the duration of disease. Facial atopy and allergic keratoconjunctivitis (AKC) related to the metaplasia of the ocular surface (P < 0.001). Patients with reduced goblet cell density also showed low BUT levels (P < 0.001). CONCLUSION: Ocular surface disorder of AD characterized by goblet cell loss and conjunctival squamous metaplasia seemed to evolve independently of the duration of disease but worsen with increased number of flare-ups. Direct epithelial damage by the allergic reaction, disorder of tear quality, and quantity may be important in the genesis of the atopic ocular surface disease.     

Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry

In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold has bound to the antigens in the cells. We investigated several aspects of gum arabic-silver lactate-hydroquinone enhancement solution (Danscher solution) by examining gold-conjugated antibodies embedded in agar, sectioned on a vibrotome, and enhanced with different solutions. The rate of silver enhancement was optimized in 50% gum arabic and 200 mM HEPES buffer, pH 5.8. We also examined chemicals used as developers and found that N-propyl gallate (NPG) gave a more uniform development than the routinely used hydroquinone (HQ). The diameter of the silver-enhanced particles after incubation in osmium tetratoxide (OSO4) decreased somewhat with longer incubation time and higher percentages, but the density (number per unit area) of silver-enhanced particles was little changed. The loss of silver-enhanced particle diameter was reduced by lowering the concentration of OSO4 to 0.1%. Comparison of commercial small gold probes showed that NPG enhancement of Nanogold gave more uniform particle size and a better correlation between enhancement time and particle density. When this procedure was applied to cell cultures with monoclonal antibodies, the silver-enhanced particles were similar to those in the agar sections. When free-floating tissue sections were used, longer silver enhancement times were needed to obtain similarly sized particles. This new NPG-silver-enhancement procedure offers a reliable and easy method to localize proteins in cultured cells and tissue sections by pre-embedding electron microscopic immunocytochemistry.     

Neuroendocrine carcinomas: role of immunocytochemistry and electron microscopy

Neuroendocrine tumors occur in many sites of the body and can present significant diagnostic problems when poorly differentiated. To identify a tumor as neuroendocrine, pathologists commonly use either immunocytochemistry or electron microscopy. In this report, the various immunocytochemical reagents are reviewed along with the ultrastructural features of neuroendocrine tumors. Site-specific variations in neuroendocrine tumors are discussed. A cost-effectiveness evaluation was performed on tumors from one laboratory which showed that electron microscopy was a less expensive diagnostic modality if more than three antibodies were necessary to arrive at the correct pathological diagnosis.