Cytocentrifuged Specimens Research Articles

Exfoliative Cytology and Cytocentrifuge Preparation of Oral Premalignant and Malignant Lesions

Objective: Oral exfoliative cytology is a sensitive and reliable diagnostic tool for the early diagnosis of oral cancer. Traditional exfoliative cytology, though popular, has limitations which could be overcome using cytocentrifuge preparations. The purpose of this study was to compare the efficacy of cytocentrifuged specimens with that of conventionally prepared smears in the diagnosis of oral premalignancy and malignancy. Study Design: Exfoliative cytology was performed on 30 patients with oral premalignant and malignant lesions. The yield was smeared immediately onto a slide for conventional exfoliative cytology (group A). The lesion was then scraped again and the yield was suspended in a bottle containing a fixative solution for cytocentrifuge apparatus-assisted smearing (group B). Both smears were stained with rapid Papanicolaou stain and assessed for cellularity, cellular and nuclear morphology, evaluation of mitosis and background. Result: All the criteria assessed were statistically significant (p value of <0.001) in group B except for mitosis, which was significant in group A. Conclusion: Cytocentrifuge preparations can preserve the cellular details and reduce the overlapping of cells, enabling precise interpretation and thus aiding in the diagnosis of premalignant and malignant lesions of the oral cavity.

Prevalence and significance of extracellular myelin‐like material in canine cerebrospinal fluid

Myelin-like material in canine cerebrospinal fluid (CSF) specimens has been attributed to demyelinating or myelomalacic conditions. In our experience, myelin-like material is observed frequently, especially in lumbar samples, and in a variety of disease conditions. The objective of this study was to determine if there are associations between the presence of myelin-like material and CSF collection site, body weight, underlying disease, and patient outcome. Wright-Giemsa-stained cytocentrifuged specimens of CSF from the cerebellomedullary cistern (n=51) and lumbar cistern (n=47) of 98 dogs with neurologic disease were evaluated retrospectively for the presence and amount of extracellular myelin-like material. Results were compared based on collection site, body weight, type of neurologic disease, and outcome. Myelin-like material was observed in 20/98 (20%) samples and was more frequently observed in lumbar (17/47, 36%) than cerebellomedullary samples (3/51, 6%) (P=.0028). Samples from dogs <10 kg were more likely to contain myelin (14/36, 39%) compared with dogs > or =10 kg (5/60, 8%) (P=.0052). Larger amounts of myelin-like material were observed in CSF from dogs with intervertebral disk disease compared with other diseases (P=.045). No association was found between myelin-like material and outcome. The association of extracellular myelin-like material in canine CSF samples with sampling site and body weight suggests it is more often an artifact of collection technique and anatomy rather than the result of neurologic disease. Myelin-like material in CSF is not associated with a poorer prognosis.

The clinical significance of eosinophils in the amniotic fluid in preterm labor

Objective. White blood cells are not traditionally considered to be normally present in amniotic fluid. This study was conducted after the observation that a patient with preterm labor and intact membranes had eosinophils as a predominant cell in the amniotic fluid, and had an episode of asthma during the index pregnancy. The goal of this study was to determine whether women presenting with preterm labor with eosinophils in the amniotic fluid had a different outcome than those without eosinophils as the predominant white blood cell in the amniotic cavity.Methods. This retrospective case–control study included women who presented with preterm labor and intact membranes between 24 and 34 weeks of gestation. Patients underwent an amniocentesis shortly after admission for the assessment of the microbiologic status of the amniotic cavity and/or fetal lung maturity. Amniotic fluid was cultured for aerobic and anaerobic bacteria as well as genital mycoplasmas. Cytologic studies included amniotic fluid white blood cell count and differential, which was performed on cytocentrifuged specimens. Patients with microbial invasion of the amniotic cavity and/or an amniotic fluid white blood cell count >20 cells/mm3 were excluded from the study. Cases were defined as women in whom the differential contained >20% of eosinophils. Controls were selected among women with an amniotic fluid eosinophil count ≤20% and matched for gestational age at amniocentesis. The analysis was conducted with non-parametric statistics.Results. The study population consisted of 10 cases and 50 controls. Gestational age and cervical dilatation at admission were similar in both groups. Cases had a lower gestational age at delivery than controls [34.6 weeks, inter-quartile range (IQR) 32–37.3 weeks vs. 38.0 weeks, IQR 35–40 weeks, respectively; p = 0.018]. The prevalence of preterm delivery ≤35 weeks was higher among patients who had >20% eosinophils than in the control group [50% (5/10) vs. 18% (9/50), respectively; p = 0.029]. Similar results were observed for delivery at <37 weeks [cases: 70% (7/10) vs. controls: 36% (18/50); p = 0.046].Conclusions. Women with preterm labor and intact membranes who have a large proportion of eosinophils in the amniotic fluid are at an increased risk for spontaneous preterm delivery. These patients may have had an episode of preterm labor related to a type I hypersensitivity reaction.

Cerebrospinal fluid from a dog with hind limb ataxia

A 9-year-old spayed female German Shepherd dog with a history of orthopedic disease was presented to the North Carolina State University Veterinary Teaching Hospital for evaluation of recent, progressive, bilateral, hindlimb ataxia. Analysis of cisternal and lumbar cerebrospinal fluid (CSF) samples revealed normal total nucleated cell counts and a mild increase in protein concentration in the lumbar sample. In cytocentrifuged specimens of both CSF samples, aggregates of refractile, angular to irregular, pale blue to colorless, crystalline material were observed in the background. Some of the material appeared birefringent under polarized light. Differentials for the material included contrast agent, epidural anesthetics or other pharmacologic agents, or artifact introduced through sample processing, collection, or handling. Based on investigation of clinical and laboratory processes it was determined that tubes used to collect CSF in the hospital recently had been changed from additive-free glass tubes to silica-coated shatter-resistant plastic tubes (BD Vacutainer Plus serum tubes, silicone-coated, Becton Dickinson). A cytocentrifuged preparation of saline placed in a silica-coated tube contained crystalline material identical to that observed in the CSF samples; saline placed in an additive-free glass tube contained no material. In this case, we document the microscopic appearance of highly concentrated silica particles in cytocentrifuged preparations of CSF and underscore the importance of recognizing and identifying this artifact in cytologic preparations.

Proteomic evaluation of archival cytologic material using SELDI affinity mass spectrometry: potential for diagnostic applications.

Proteomic studies of cells via surface-enhanced laser desorption/ionization spectrometry (SELDI) analysis have enabled rapid, reproducible protein profiling directly from crude samples. We applied this technique to archival cytology material to determine whether distinct, reproducible protein fingerprints could be identifiedfor potential diagnostic purposes in blinded specimens. Rapid Romanowsky-stained cytocentrifuged specimens from fine-needle aspirates of metastatic malignant melanoma (with both known cutaneous primary and unknown primary sites), clear cell sarcoma, and renal cell carcinoma and reactive effusions were examined using the SELDI technology. A unique characteristic fingerprint was identified for each disease entity. Fifteen "blinded" unknown samples then were analyzed. When the protein profilefingerprints were plotted against the known fingerprints for the aforementioned diagnoses, the appropriate match or diagnosis was obtained in 13 (87%) of 15 cases. These preliminary findings suggest a substantial potential for SELDI applications to specific pathologic diagnoses.

Immunocytologic staining of cytokeratin in bone marrow aspirates to detect micrometastatic cells fails in patients with metastatic urologic carcinoma

The detection of cytokeratin-positive cells in bone marrow of patients with various carcinomas is considered an adverse marker. To establish a routine diagnostic procedure to detect these cells in patients with urologic carcinoma, we evaluated different immunocytological techniques in parallel. The study included 50 patients with urologic carcinoma (16 with metastatic disease) and 10 healthy volunteers. Bone marrow was aspirated from the iliac crest; after density centrifugation, cytocentrifuged specimens were stained according to optimized modifications of conventional immunocytologic techniques. Overall only 3% of patients with locally restricted carcinoma and only 19% with metastatic disease showed cytokeratin-positive bone marrow cells. Nonspecific labeling of bone marrow cells in the immunoglobulin isotype controls was a major problem, demonstrating the need for numerous detailed controls. If all nonspecific reactions were excluded, up to 20% of the healthy controls yielded positive results as well. The presence of cytokeratin-positive cells in bone marrow aspirates shows only slight correlation with systemic metastasis. The biologic and clinical relevance of this immunocytologic marker must be judged cautiously.

Intraocular lymphoma. Immunopathologic analysis of vitreous biopsy specimens.

Immunologic analysis of cell surface markers (immunophenotyping) has become a standard procedure in the evaluation of systemic lymphomas. However, attempts to apply these techniques to intraocular lymphoma have not been uniformly successful. We successfully immunophenotyped five consecutive cases of intraocular lymphoma using immunoperoxidase surface marker analysis of cytocentrifuged specimens in two cases and flow cytometry in three. In all five cases, a monoclonal B-cell population was unequivocally present. Contrary to previous reports, we found surface marker analysis of vitreous biopsy specimens to be helpful in the diagnosis and treatment of intraocular lymphoma. Not only did it support the cytologic diagnosis but it allowed comparison of the immunophenotype of vitreous infiltrates with that of previous or subsequent lymphomatous lesions from nonocular sites.

A fast and reliable system for microcomputerized DNA cytofluorometry in tumour pathology.

A microcomputerized cytofluorometry system based on a Leitz MPV 3 cytophotometer, and intended for DNA measurements in tumour pathology is described. The system has been equipped with a reference channel for correction of excitation light instability. The importance of the adjustment of the epi-illumination for optimal performance of the reference channel is stressed, and a detailed description is provided. An apparatus variability well below 1% CV is obtained even during periods of marked instability of the arc-lamp. Software for conventional cytofluorometry with which about 200 cells can be measured in 20 min is presented. In addition a measuring technique where cells are not positioned, but are just passed through the excitation light spot is described. Preliminary results on cytocentrifuged specimens of human cancers indicate that this system is at least three times faster than conventional fluorometry. The increased speed of measurements considerably extends the possibilities to evaluate cell proliferation using static cytofluorometry. The measuring capacity of course is dependent upon the quality of the specimen. In well prepared cytocentrifuged specimens 1000 cells have been measured in 35 min.

Bactericidal mechanisms of human breast milk leukocytes.

The functional capacity of human breast milk phagocytes was evaluated with both bactericidal and biochemical assays. Acridine orange was used as a vital stain for bacteria to directly visualize phagocytosis and killing. Bactericidal capabilities were further examined by colony count and chemiluminescent methods. Cytocentrifuged specimens stained for myeloperoxidase exhibited enzyme activity in breast milk leukocytes equal to that of peripheral neutrophils. A radioisotopic assay of hexose monophosphate shunt activity demonstrated metabolic activity in breast milk leukocytes greater than that in peripheral blood neutrophils. However, the chemiluminescent response of breast cells was negligible, apparently the result of quenching secondary to fat present in the milk; preincubation of human blood leukocytes with the fatty layer of breast milk produced similar inhibition in the chemiluminescence assay. By most parameters breast milk phagocytes are at least equal to blood neutrophils.

PMNs in normal spinal fluid examined by the cytocentrifuge technique.

The presence in unconcentrated CSF specimens of even a single polymorphonuclear (PMN) cell has been regarded as diagnostic of central nervous system (CNS) disease. In 18 of 50 consecutive cytocentrifuged specimens obtained from patients without evidence of CNS disease, however, we found 1 to 6 PMN cells. The observation of occasional PMN cells in concentrated CSF specimens does not appear to be diagnostic of a CNS abnormality.

Differential Count of Urinary Leukocytes and Renal Epithelial Cells

Urine samples from 143 patients were treated in two ways. One part of every sample was centrifuged in a conventional centrifuge and the other one in the Cytospin(R) centrifuge, a so-called cytocentrifuge. Cytocentrifligation seemed to be better suited for cell concentration than common centrifugation, yielding a greater number of specimens with a sufficient number of cells for a differential count. The sediments obtained by common centrifugation were examined by phase contrast microscopy, while the cytocentrifuged specimens were examined by conventional light microscopy after fixation and staining with Papanicolaou technique. By both methods were determined the percentages of renal epithelial cells, granulocytes and mononuclear leukocytes. Similar results were obtained by both methods. The percentages of renal epithelial cells and granulocytes varied from case to case. The percentage of mononuclear leukocytes was small in all cases. Use of fixation and staining seemed to enhance the possibilities of identification of histiocytes and lymphocytes.