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Uterine lavage (UL) is a routine diagnostic procedure for endometritis. In UL the fluid is centrifuged and the sediment smeared. Samples prepared in cytocentrifuges, the so-called “cytospins”, are useful for evaluating cells in fluids, but never been used in UL. The aim of this study was to assess the usefulness of cytospins after UL, comparing automatic versus manual cytocentrifuges, and to determine their value for the diagnosis of endometritis. The study was divided in two parts. Firstly, UL was performed in 16 mares and a small part of the retrieved fluid was cytocentrifuged in an automatic (PreCyto) and manual (PreMan) cytocentrifuge, whereas the remaining fluid was centrifuged. After that, the sediment was divided into three quotas. One quota was smeared, one was processed in an automatic cytocentrifuge (PostCyto) and the last quota was cytospinned in a manual apparatus (PostMan). Cytospins obtained were scored for cellularity, cell preservation, presence of inflammatory cells, bacteria and contaminants; results were compared with sediment smears. Secondly in this study, the best cytospin method was compared with sediment smears in another group of 13 mares, which had endometrial biopsy after UL. Agreement between sediment smears and cytospins was poor to moderate. Compared to sediment smears, cytospins were more cellular, with better morphological details. Urine crystals and fecal contamination were detected more often in cytospins (especially PostCyto and PostMan). No differences in the percentage of inflammatory or epithelial cells existed. PostMan was considered the best method to evaluate UL fluid and it had higher sensitivity (80%), compared with sediment smears (60%), for diagnosing endometritis. Cytocentrifugation offers significant advantages over sediment smears and the manual cytocentrifuge is well suited for horse stable conditions.

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

Suspension cells can be prepared for staining by several different methods. A simple method for detecting intracellular antigens in cells that grow in suspension is to attach the cells to a solid substrate before fixation. This can be achieved by the use of a cytocentrifuge. For surface staining, suspension cells can be attached to slides by cross-linking with poly-l-lysine. Lysine can be polymerized to any desired length, and poly-l-lysine will bind to most solid supports through its charged side chains. The positively charged polymer will provide a site for binding of cells (which carry an overall negative charge). Although this cross-link is not covalent, it is sufficiently strong for most cell-staining techniques.

Background: Cytological examination of pericardial effusion fluids is important in diagnosing the etiology of underlying disease, staging, and prognosis of cancer. Aims and Objectives: (1) To study cytological evaluation of pericardial effusions in various pathological conditions in a tertiary care center. (2) To analyze their frequency and clincopathological correlation of the diagnosis. Materials and Methods: Our study was a retrospective study performed in the Department of Pathology from 1st January 2012 to 31st December 2016. The study sample included all the pericardial effusions submitted in the pathology department for cytological evaluation. Clinical details and relevant parameters correlated with clinical findings. Each fluid underwent cytospin and cytocentrifuge along with preparation of conventional smears. Results: Of 120 cases, 80% were of benign effusion and 20% were of malignant effusion. Male-to-female ratio was 1.44:1 with patient age ranging from 3 to 90 years. Conclusion: Benign effusions can been seen in younger age group and malignant ones in the older age group. The preliminary pericardial fluid analysis in resource-limited settings is the most convenient and cost-effective method for accurate diagnosis. It reduces the demand of invasive investigations and its complications. At times, it is the first test to point toward underlying malignant process thereby affecting the prognosis, survival, and treatment outcome of the patient.

Background: Cytologic examination of body fluids commonly involves the use of direct or sediment smears, cytocentrifuge preparations, membrane filter preparations, or cell block sections. Cytospin and cell block techniques are extremely useful in improving cell yield of thin serous effusions and urine samples, and ensure high diagnostic efficacy. Materials and Methods: We studied cytospin preparations and cell block sections prepared from 180 samples of body fluids and urine samples to compare the relative efficiency of cell retrieval, preservation of cell morphology, ease of application of special stains, and diagnostic efficacy. Samples were collected and processed to prepare cytospin smears and cell block sections. Results: We observed that overall, cell yield and preservation of individual cell morphology were better in cytospin preparations as compared to cell blocks, while preservation of architectural pattern was better in cell block sections. The number of suspicious cases also decreased on cell block sections, with increased detection of malignancy. It was difficult to prepare cell blocks from urine samples due to low cellularity. Conclusions: Cytospin technology is a quick, efficient, and cost-effective method of increasing cell yield in hypocellular samples, with better preservation of cell morphology. Cell blocks are better prepared from high cellularity fluids; however, tissue architecture is better studied, with improved rate of diagnosis and decrease in ambiguous results. Numerous sections can be prepared from a small amount of material. Special stains and immunochemical stains can be easily applied to cell blocks. It also provides a source of archival material.

Cytocentrifuge slide (“cytospin”) is a simple and rapid technique that is mainly used in the cytology laboratory. We have routinely used the technique in flow cytometry (FCM) laboratory at Montefiore Medical Center for more than 15 years. Our aim is to illustrate the contribution of cytospin in direct cellular morphologic visualization in FCM immunophenotyping, in result interpretation, and additionally as a quality control tool. Four thousand two hundred forty-eight cases sent to our FCM Laboratory in the past 21 months were studied retrospectively. The clinical history, cytospins, immunophenotyping panels, FCM contour plots, final FCM reports, and pathology reports were reviewed. Morphologic examination of the cytospin is a cost-effective tool for sample quality screening. Of the 4248 immunophenotyping cases, 359 (8.4%) cases were canceled, according to laboratory protocol based on cytospin review. Morphologic analysis of the cytospin provides extra information that guides both selection of the FCM immunophenotype panel and gating the cells of interest. Additionally, morphologic analysis of cytospin can yield early diagnosis when cohesive benign or malignant non-hematologic cells are present or even infectious organisms such as parasites are identified. Finally, the cytospin is a convenient tool for morphology-phenotype correlation. Cytospin is cost-effective and provides important morphologic information that facilitates FCM immunophenotyping panel selection and result interpretation.

Separation and detection of exfoliated tumor cells (ETCs) from bronchoalveolar lavage fluid (BALF), namely the liquid biopsy of BALF, has been proved to be a valuable tool for the diagnosis of lung cancer. Herein, we established a rapid liquid biopsy of BALF based on a dual-layer PERFECT (precise, efficient, rapid, flexible, easy-to-operate, controllable and thin) filter system for the first time. Methods: The dual-layer PERFECT filter system consists of an upper-layer filter with large micropores (feature size of 49.4 ± 0.5 μm) and a lower-layer filter with small micropores (9.1 ± 0.1 μm). The upper-layer filter contributes to the isolation of cell clusters and removal of mucus from BALF samples, meanwhile the lower-layer one targets for the separation of single ETCs. First, separation of 10000 spiked A549s (cultured lung cancer cells) from 10 mL clinical BALF samples (n=3) were performed to investigate the performance of the proposed system in rare cell separation. Furthermore, separation and detection of ETCs and ETC clusters from clinical BALF samples were performed with this system to test its efficacy and compared with the routine cytocentrifuge. The clinical BALF samples were collected from 33 lung cancer-suspected patients with visible lesions under bronchoscope. The final histopathological results showed that 20 samples were from lung cancer positive patients while the other 13 cases were from lung cancer negative patients. Results: The recovery rate of spiked A549 cells from clinical BALF samples with the developed system (89.8 ± 5.2%) is significantly higher than that with the cytocentrifuge (13.6 ± 7.8%). In the preliminary clinical trial, although 33 clinical BALF samples with volume ranging from 6 mL to 18 mL showed greatly varied turbidity, filtrations could be finished within 3 min for 54.6% of samples (18/33), and 10 min at most for the rest. The dual-layer PERFECT filter system is proved to have a much higher sensitivity (80.0%, 95% CI: 55.7%-93.4%) than the routine cytocentrifuge (45.0%, 95% CI: 23.8%-68.0%), p=0.016 (McNemar test, two-tail). Moreover, the sensitivity of this platform is neither interfered by the variations of turbidity of the BALF samples, nor associated with the types of lung cancer. Conclusions: The easy and rapid processing of BALF samples with varying volume and turbidity, competitive sensitivity and good versatility for different lung cancer types will make the established dual-layer PERFECT filter system a promising approach for the liquid biopsy of BALF. The high-performance BALF-based liquid biopsy will improve the cytopathological identification and diagnosis of lung cancer.

Objective: The history of Sayk's cell sedimentation chamber is examined and reviewed in the context of clinically utilizable cerebrospinal fluid (CSF) cytology. Methods: A review of the literature was undertaken in PubMed and Google Scholar to search for primary and secondary sources on the history of CSF diagnosis. Moreover, documents in the archives of the Universities of Rostock and Jena, the Brain Research Institute of Cécile and Oskar Vogt in Düsseldorf and the Centre for Neurology at Rostock University were used. Results: The cell sedimentation chamber, developed by Johannes Sayk (1923-2005) at the beginning of the 1950s, enabled, for the first time, CSF cells to be presented with a quality comparable to the blood profile, with a low technological expenditure and using all staining methods suitable for haematological cells. The procedure could be performed quickly and cost efficiently, and therefore, it was easy to integrate it into clinical routine and it quickly became a widely used procedure. In Europe, it was considered the standard method for cytodiagnosis of CSF until the 1990s. Conclusions: The procedure revolutionised CSF diagnostics and paved the way for modern qualitative CSF cytology. In course of time, the cell sedimentation chamber was superseded by the cytocentrifuge.

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 5 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n=10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Background At diagnosis, there are prognostic implications of low-level leukemic blasts (CNS 2) in the cerebrospinal fluid (CSF) of patients with acute lymphoblastic leukemia (ALL). However, the significance of post-induction CNS 2 results and the impact of equipment on CNS 2 prevalence have not been well studied. Procedure A single-institution retrospective cohort study was conducted to analyze the outcome of patients with ≥1 post-induction CNS 2. A subanalysis compared the proportion of CNS 2 CSF results using 2 different cytocentrifuges; the Shandon Cytospin used from 2005 to 2008 and the Wescor Cytopro used from 2010 to 2014. Results Over 4500 post-induction CSF samples were analyzed, of which 59 were CNS 2. In covariate-adjusted analyses, post-induction CNS 2 did not significantly increase relapse risk. The proportion of CNS 2 results increased 4.3-fold in noninfants and 6.3-fold in infants using the Wescor Cytopro. Cytocentrifuge machine did not affect CNS 3 prevalence. Conclusions These findings support our current practice of not changing management based on a post-induction CNS 2 CSF and highlight how equipment changes can significantly influence testing results. More data are needed to analyze relapse by subpopulations, such as those with repeated CNS 2 findings.

Objective: Direct microscopic visualization is the most specific method for detecting intestinal parasites and is commonly achieved by stool examination or mucosal biopsy. However, postfixation, the intestinal biopsy fragment is often curled, and the entire surface of the biopsied mucosa is seldom viewed microscopically. Tissue processing further distorts morphology of the organisms and causes diagnostic difficulties. Examining multiple sections for parasite detection is time-consuming and often requires aid of special stains and/or immunohistochemistry. To overcome these disadvantages, we hypothesized that the fixative in which biopsies are transferred may provide a valid representation of the biopsied mucosal surface and therefore aid in the identification of mucosal surface parasites.Materials and Methods: Formalin in which biopsies were transferred was retained, stored at 4°C and processed with a cytocentrifuge. Totally, 120 consequent duodenal biopsy fixatives were processed in this way and the cytocentrifuged smears visualized after May-Grunwald-Giemsa staining. Findings of these smears were correlated with their corresponding formalin fixed paraffin embedded tissue sections.Results: Cytocentrifuged formalin preparations were found to be representative of the mucosal surface contents. Giardia trophozoites were visualized in 10/120 preparations with distinct morphological characteristics which were seldom appreciable in tissue sections, eliminating the need for special stains. Furthermore, two of the corresponding histology sections did not demonstrate the parasites despite step sections, while in one case few parasites could be identified in the step sections. Conclusions: Cytocentrifuged fixative preparation is a simple and cost-effective technique which can be routinely employed for intestinal parasite characterization.

Background-The cytological examinations of serous effusions have been well accepted, and a positive diagnosis is often considered as a definitive diagnosis. It helps in staging, prognosis and management of patients of malignancies, and also give information about various inflammatory and non inflammatory lesions. Diagnostic problem arises in everyday practice to differentiate reactive atypical mesothelial cells from malignant cells by routine conventional smear method. Aim- To compare the morphological features of the conventional smear method with those of cytospin(cytocentrifuge) and cell block and also to assess the utility and sensitivity of cytospin and cell block methods in the cytodiagnosis of effusion. Material and method- One hundred and fifty samples were subjected to diagnostic evaluation. Along with conventional smear, fluids were subjected to cytocentrifuge and cell block technique. Cell blocks were prepared using 10%formal alcohol as a fixative agent. Smears obtained by each of techniques were scored for different parameters. Statistical analysis with Wilcoxon rank sum test was performed and also Index of Qualitative Variation(IQV) was calculated to compare the results obtained by each of the above three methods. Results- Cellularity and additional yield for malignancy was more for Cytospin and Cell Block technique. Conclusion- The cytospin and cell block method provide high cellularity, better architectural patterns, morphological features and an additional yield of malignant cells, and thereby, increases the sensitivity of the cytodiagnosis when compared to conventional smear method.

Background Smears prepared by cytocentrifugation, the so-called cytospins, are widely used in human and veterinary cytology. However, the high cost has hampered the availability of commercial cytospin centrifuges in some veterinary clinics and laboratories. Nevertheless, cytospins are important for evaluating fluids with very low cellularity such as cerebrospinal fluid (CSF) or bronchoalveolar lavage fluid (BALF). Objectives The aim of this study was to devise and test the use of a low-cost, in-house manual cytocentrifuge to obtain cytospin preparations. Methods Twenty-two fluid samples (including CSF and BALF) were collected from dogs and cats. These were processed in a conventional cytocentrifuge and in an in-house, manual centrifuge (salad spinner). The cytospins obtained by the 2 methods were compared by scoring cellularity, number of cells per field, hemodilution, cell preservation, and proportion of ruptured cells. Additionally, cell number and size were compared by morphometry. Differences between the automated and manual method were statistically assessed. Results The morphology and cellular detail of cytospin preparations produced by both methods were identical. There was an almost perfect agreement for cellularity, number of cells per HPF, hemodilution and cell preservation (kappa ≥ 0.85), and a moderate agreement for the amount of ruptured cells. Cell recovery was comparable (including in CSF and BALF). Conclusions The manual cytocentrifuge produced cytospins with similar cell yield as the automated cytocentrifuge. Considering the low cost and portability, this new method should be particularly useful for cytologic diagnosis in small clinics, developing countries, and in field studies.

Ferroptosis is an iron-dependent form of non-apoptotic cell death. Cellular energy depletion is not observed in ferroptosis. This process is characterized by the iron-dependent accumulation of lethal lipid reactive oxygen species. Cytochemical analysis of alveolar macrophages (AM) was performed in nonsmokers, control smokers and smokers with non-small-cell lung cancer (NSCLC) by light microscopy, using indexing and scorring method. After bronchoalveolar lavage (BAL) fluid separation, cytocentrifuge preparations have been made on glass slides, air dried and then stained with May-Grunwald-Giemsa for differential cell counting and by standard method for the semiquantitative estimation of cytochemical reaction positivity of AM for: chloroacetate esterase (CHL) and Perls9 reaction (for estimation of intracellular iron). At the same time, TUNEL in situ cytochemical method was used for detection of apoptosis. There was significant decrease of percentage of AM in NSCLC in comparison with nonsmokers (p

The aim of the study was to isolate and establish an Anaplasma marginale strain from Brazilian brown brocket deer, Mazama gouazoubira, in the Ixodes scapularis cell line IDE8. Blood from a free-living adult female M. gouazoubira naturally infected with A. marginale (MGI5) was inoculated intravenously into a splenectomized calf. When A. marginale rickettsemia was 2.5%, blood was collected and cryopreserved in liquid nitrogen with dimethylsulfoxide (DMSO). IDE8 cell cultures were infected with calf blood inoculated with the A. marginale (MG15) isolate. The cultures were monitored by examination of Giemsa-stained cytocentrifuge smears. Light microscopy of stained IDE8 samples revealed the first inclusions of A. marginale (MGI5) at 48days post-inoculation (d.p.i). The IDE8-infected cells contained parasitophorous vacuoles with amorphous material and a few cocci-like organisms. A sample from IDE8-infected cells from the 16th subculture (336 d.p.i.) was analyzed by nPCR, nucleotide sequencing, electron microscopy, and an indirect fluorescent antibody test (IFAT). The IFAT highlighted some IDE8-infected cells with intense fluorescence in the parasitophorous vacuole, while in other cells, fluorescence was observed only at the periphery. DNA from a culture of the MG15 isolate was amplified with A. marginale msp4 gene primers, and nucleotide sequencing of the PCR product and BLAST software analysis further confirmed 100% identity with the MGI5 blood isolate (GenBank no. JN022558.1). Electron microscopy revealed increased numbers of lysosomes in the cytoplasm of IDE8 cells. Several cells exhibited large vacuoles containing cellular debris and amorphous material. After the 29th subculture, it was not possible to detect compatible Anaplasma structures by light microscopy, and subculture samples tested negative in nPCR. Despite the failure of the attempt to establish A. marginale (MGI5) in IDE8 cells, the results demonstrated the isolate's ability to infect, survive and multiply, although in limited numbers, in IDE8 cells.

Staging and monitoring of pediatric acute lymphoblastic leukemia (ALL) includes examination of the cerebrospinal fluid (CSF). At our institution, we noted an increased incidence of low-level leukemic blasts in CSF samples from patients with ALL. This increase coincided with a conversion from the Shandon CytoSpin 4 (Thermo Fisher Scientific, Waltham, MA) to the Wescor Cytopro Rotor AC-060 (Wescor, Logan, UT). This study directly compared these 2 machines using patient samples and known concentrations of cultured leukemia cells. With patient samples, the Wescor Cytopro led to a 5- to 9-fold increase in the number of cells on a slide compared with the Shandon CytoSpin; furthermore, leukemic blasts were detected only with the Wescor Cytopro in 2 cases. Similar findings were observed using cultured leukemia cells. Thus, the detection of blasts in CSF is highly instrument-dependent. The newer, more sensitive cytocentrifuge machines identify blasts that were previously missed by older machines, but the clinical significance remains under investigation. The 5-year survival rate of pediatric patients diagnosed with acute lymphoblastic leukemia (ALL) has increased from 54.1% in the 1975-1977 period to 85.1% in the 1999-2005 period 1 because of numerous treatment advances. Despite this progress, approximately 1 in 5 patients will have a recurrence of ALL, including some as isolated central nervous system (CNS) relapses. High cerebrospinal fluid (CSF) blast count at initial diagnosis (CNS 3, cell count ≥5 WBCs/μL with blasts present) is correlated with a poorer prognosis. However, the prognostic significance of a lower blast count in the CSF (CNS 2a, cell count

Both cytoplasmic and surface-membrane antigens of human spermatozoa were detected by means of monoclonal antibodies (MoAbs) and of the alkaline phosphatase anti-alkaline phosphatase- (APAAP-) technique. Several advantages of this technique for the identification of sperm could be demonstrated. The labeling of cytocentrifuge preparations from 16 ejaculates proved the presence of glycosphingolipids, nuclear and mitochondrial antigens of spermatozoa. However, there were no HLA-molecules and other leukocyte antigens on sperm cells.

Mass spectrometry imaging is a promising technique for measuring drugs and drug metabolites in cells and tissues. In this manuscript we describe a method for the imaging of HIV protease inhibitors. As a model system we used Mono Mac 6 cells cultured with the HIV protease inhibitors saquinavir and nelfinavir deposited on glass slides using a cytocentrifuge. A sublimation/deposition device for homogeneous matrix deposition was constructed which allows imaging of these HIV protease inhibitors at clinically relevant concentrations. Using this matrix sublimation/deposition method, glass slides containing the cytocentrifuged cells can be measured and analyzed by two types of mass spectrometry techniques, viz. matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and MALDI Fourier transform ion cyclotron resonance (FTICR), and this makes it possible to perform imaging rapidly (MALDI-TOF) and with a very high selectivity (MALDI-FTICR).

Objective To develop a simplified technique for processing hemorrhagic body fluids, allowing elimination of red blood cells (RBCs) to obtain tumor cell-rich cytosmears for accurate diagnosis of malignancy. Study Design Hemorrhagic fluid is collected with ethylenediamine tetraacetic acid anticoagulant. The cells are separated by centrifugation in glass capillaries into 3 layers, with the uppermost containing supernatant, middle buffy coat and lowermost dark layer of RBCs. The smears of the buffy coat are prepared by breaking the capillaries at the junction of buffy coat and RBC layer. The procedure is named capillary centrifugation technique. This procedure was developed in the cytology laboratory at King George's Medical College, Lucknow, India in 1974. Results Cell yield is good and cellular details are well preserved. No cell debris is present on the slides. The background of the smears is absolutely clear. Any number of slides may be prepared for special study for exact typing of tumor cells. Conclusion This is a simple, economical procedure and a good substitute for a cytocentrifuge machine for preparing smears from small amount of fluids. Technicians learn it quickly and are quite comfortable with the procedure.

The vital staining method using prestained slides permits satisfactory characterization and differentiation of benign and malignant cells present in CSF using 10 and 20 mul of CSF. In comparison the sedimentation technique of Sayk and the cytocentrifuge with subsequent Pappenheim staining require larger volumes of CSF. They have the disadvantage of possible loss of cells and of cell denaturation.

Objective To define how to best handle cerebrospinal fluid (CSF) specimens to obtain the highest positivity rate for the diagnosis of malignancy, comparing two different methods of cell concentration, sedimentation and cytocentrifugation. Methods A retrospective analysis of 411 CSF reports. Results This is a descriptive comparative study. The positive identification of malignant CSF cells was higher using the centrifuge than that using the Suta chamber (27.8% vs. 19.0%, respectively; p = 0.038). Centrifuge positively identified higher numbers of malignant cells in samples with a normal concentration of white blood cells (WBCs) (< 5 cells/mm3) and with more than 200 cells/mm3, although this was not statistically significant. There was no lymphocyte loss using either method. Conclusions Cytocentrifugation positively identified a greater number of malignant cells in the CSF than cytosedimentation with the Suta chamber. However, there was no difference between the methods when the WBC counts were within the normal range. RESUMO Objetivo Definir qual a melhor forma de concentrar amostras de LCR para obter maior porcentagem de positividade para o diagnóstico de infiltração neoplásica. comparando dois métodos diferentes de concentração de células, sedimentação e citocentrifugação. Métodos Análise retrospectiva de 411 laudos de LCR. Resultados Estudo comparativo descritivo. A identificação de células neoplásicas no LCR foi mais elevada quando usada a citocentrífuga do que a câmara de Suta (28% vs 19,0%, respectivamente; p = 0,038). Centrifugação identificou maior número de células neoplásicas em amostras com concentração de células < 5 células/mm3 e superior a 200 células/mm3, embora não significativo. Não houve perda de linfócitos usando qualquer um dos métodos. Conclusões A citocentrifugação identificou um número maior de células malignas no LCR do que a sedimentação com a câmara de Suta. No entanto, não houve diferença entre os métodos quando as contagens de leucócitos estavam dentro do intervalo normal.

Objetivo: estandarizar la técnica de citología en base líquida mediante el uso del medio fijador (BDSUREPAth) y de citocentrífuga e identificar las posibles ventajas sobre citología convencional en muestra compartida de tomas cervicouterinas. Métodos: se incluyeron 92 muestras de mujeres que asistieron a las campañas del programa de proyección social de la Fundación Universitaria de Ciencias de la Salud, Bogotá DC, Colombia. Resultados: de las 92 muestras, 89 fueron negativas para citología convencional y 75 para base líquida, observándose un mayor número de casos con anormalidades en células escamosas en citología en base líquida. Conclusiones: las células escamosas mantienen su tonalidad citoplasmática siendo basófilas o acidófilas, conservando lo translúcido de los citoplasmas; las células glandulares preservan su patrón en panal de abejas o empalizada. Mediante citología en base liquida se obtienen fondos más limpios, las atipias de las células epiteliales fueron más fáciles de identificar al igual que los microorganismos patógenos. Abreviaturas: VPH, virus del papiloma humano; ASC-US, células escamosas atípicas de significado indeterminado.

Background: Determining T-cell phenotypes of lung cells obtained by bronchoalveolar lavage (BAL) is frequently clinically useful, particularly for evaluating causes of interstitial lung disease. The current standard of determining CD4/CD8 T-cell subsets by immunohistochemical (IHC) staining of cytocentrifuge slides is labor-intensive and subject to interpreter variation. Flow cytometry (FCM) is a precise and rapid method commonly used in research to characterize cells in the lung. However, few studies address the methodology of analysis of BAL lymphocytes by FCM. Methods: Patients underwent bronchoscopy for clinical purposes. A BAL cell differential and T-cell subtype was requested by the treating physician to supplement the evaluation of patients with suspected interstitial lung disease. We used a commercially available T-cell antibody reagent, approved for analysis of blood via FCM, for T-cell subtyping of clinical BAL specimens. Results: The percentages of CD4 and CD8 T-cell populations, as well as the CD4/CD8 ratios showed excellent correlation with IHC staining of cytocentrifuge slides regardless of the acquisition program used, as long as the gating strategy remained consistent (r ≥ 0.9693 for CD4, r ≥ 0.9589 for CD8, and r ≥ 0.9485 for the CD4/CD8 ratio). Conclusion: These findings validate the use of standardized, commercially available antibody cocktails for BAL lymphocyte subtyping, making this technique available to clinicians and researchers with access to a three-color or four-color flow cytometer. © 2006 International Society for Analytical Cytology

Hepatocytes with 2 or more nuclei are seen at a certain percentage in the normal rat liver and show changes according to mitogenic stimuli including regeneration from injury. In the present study, analysis of nuclearity and cell kinetics of hepatocytes was conducted by combining cytocentrifuge preparations of isolated hepatocytes and bromodeoxyuridine (BrdU) cumulative labeling in 6-week-old female Sprague-Dawely rats at periods of 7, 14, and 28 days, respectively. In cytocentrifuge preparations, hepatocytes with intact cytoplasms were obtained, from which nuclearity was clearly and readily defined. The majority of hepatocytes were mononuclear or binuclear. The percentage of cells with 3 or more nuclei was very low. The percentages of each nuclearity class were similar throughout all labeling periods. The ratio of mononuclear to binuclear cells was approximately 6:4. The BrdU labeling index per total nuclei increased according to the labeling period. For the labeling index of each cell type, the number of BrdU positive mononuclear cells and binuclear cells with 2 positive nuclei (WP) increased according to the labeling period at a ratio of approximately 3:1. In the binuclear cells, WP cells and cells with 2 negative nuclei were dominant, whereas, cells with 1 positive and 1 negative nuclei were very rare. From these observations, the present method was thought to be well suited for analysis of nuclearity and cell kinetics in hepatocytes and may be useful for analysis of changes in the liver induced by various compounds.

Cell staining is a necessary and useful technique for visualizing cell morphology and structure under a microscope. This technique has been used in many areas such as cytology, hematology, oncology, histology, virology, serology, microbiology, cell biology, and immunochemistry. One of the key pieces of equipment for preparing a slide for cell staining is cytology centrifuge (cytocentrifuge) such as cytospin. However, many small labs do not have this expensive equipment and its accessory, cytoclips (also expensive relatively), which makes them difficult to study cell cytology. Here we present an alternative method for preparing a slide and cell staining in the absence of a cytocentrifuge (and cytoclips). This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining. The method presented is simple, rapid, economic, and efficient. This method may also avoid a possible change in cell morphology induced by cytocentrifuge.

OBJECTIVE: To investigate the applicability of the UItrafast Papanicolaou stain to the cytology of fluids and to compare it with other methods. STUDY DESIGN: Over a 30-month period, 528 unfixed fluids (462 serous effusions, 48 pelvic washings, 16 cyst fluids and 2 bile duct drain fluids) were mixed thoroughly and centrifuged. Two Swedish-style air-dried smears were made and stained with Diff-Quik (Mercedes Medical, Inc., Sarasota, Florida, U.S.A.) and Ultrafast Papanicolaou stain (Richard Allan Scientific, Kalamazoo, Michigan, U.S.A.), and the remaining sediment was fixed in CytoRich Red (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.), centrifuged onto a 17.5-mm circle with a Hettich cytocentrifuge and stained by the Papanicolaou method. RESULTS: For the 115 malignant fluids, Ultrafast Papanicolaou stain was the preferred method in the 94 nonhematopoietic malignant fluids, Diff-Quik was the preferred method in the 9 hematopoietic malignancies, and CytoRich Red was the preferred preparation in 8 bloody effusions containing rare cancer cells and 4 malignant pelvic washings. The diagnostic turnaround time of smears stained by Ultrafast Papanicolaou stain was

Our purpose was to demonstrate that 17.5-mm liquid-based cytocentrifuge circle-slides made using a Hettich (Andreas Hettich Co., Tuttlingen, Germany) cytocentrifuge are at least as efficacious as conventional smears for detecting cervical abnormalities. One hundred conventional smears were collected with cytobrushes and wooden spatulas from high-risk women. Both devices were then placed into CytoRich Red (AutoCyte, Inc., Burlington, NC). Cells were concentrated from CytoRich Red by centrifugation and suspended in CytoRich Yellow. Two 17.5-mm circle-slides were produced, compared to each other in order to test reproducibility of diagnoses between slides, and compared to conventional slides. Sixty-five normals, three ASCUS, three LSIL, and two HSIL matched. Overall, cytocentrifugation yielded 27 additional findings among 25 cases. Also, it downgraded one conventional ASCUS to normal (immature metaplasia and chronic inflammation) and one conventional HSIL to normal (transitional cell metaplasia). Cytocentrifugation of 14 conventional normals afforded four ASCUS and 10 LSIL. Cytocentrifugation of 11 conventional ASCUS afforded nine LSIL and two HSIL (one with AIS). One HSIL + AIS was found with a conventional HSIL. Cytobrushes and wooden spatulas can be used to collect material for liquid-based cervico-vaginal cytology if they are placed into CytoRich Red. Hettich cytocentrifuge slides were more reliable than conventional smears in presenting well-dispersed, sharply imaged cells, affording greater diagnostic sensitivity and certainty. Compared to previous split-sample studies, three different outcomes were noted: 1) the endocervical component was consistently well-represented; 2) AIS was seen more often in liquid-based slides; and 3) the liquid-based slide did not underdiagnose its conventional companion. The liquid-based preparations downgraded one ASCUS to normal and one conventional HSIL to transitional-cell metaplasia. Histology correlation of these two cases agreed with the liquid-based cytology diagnoses, which validates their specificity. These improvements are ascribed to differences in fixation and processing. The low cost, ease of operation, and reusable chambers of the Hettich cytocentrifuge make it a cost-effective liquid-based cytology instrument, especially for small to intermediate-size laboratories. The claims that this paper makes must be proven by additional studies, and test implementation needs to be scrutinized by appropriate regulatory agencies whose standards may vary from country to country. Diagn. Cytopathol. 2000;22:86-91.

During antibiotic-treated urinary tract infection in non-catheterized geriatric inpatients, bacteriuria disappeared within 1-2 days. Pyuria, on the other hand, disappeared within 4-7 days. After withdrawal of antibiotic therapy, bacteriuria reappeared quicker than pyuria. It is suggested that the time lag between bacteriuria and pyuria is due to the fact that bacteriuria reflects bacterial invasion into the urinary tract, whereas pyuria reflects the inflammatory response of the urinary tract.

Differential uterine curettage may be performed for the evaluation of abnormal uterine bleeding. The purpose of this study was to test the feasibility of substituting cytological methods for differential uterine curettage to evaluate the entire glandular axis of the uterus and the transformation zone. In order to simulate differential uterine sampling, the endocervix was brushed in 455 uteri from which an endometrial cytology sample had been previously collected. Twenty-five percent of the brushes' cell-sample was examined using two 17.5 mm circle-slides prepared with a Hettich cytocentrifuge. The resulting slides were homogeneous and cellular. They were compared to tissue sections of matched hysterectomy specimens. Endometrial adenocarcinoma was overrepresented in the endocervical cytology preparations. This was ascribed either to prior endometrial collection with "drop-down contamination" of the endocervix and/or to entry of the cytobrush into the endometrial cavity. Otherwise, cytological and histological diagnoses were comparable for squamous and endocervical-adenomatous lesions. Under the conditions of this simulation, endometrial and endocervical brushings appeared to complement each other as case-finding techniques for evaluating abnormal uterine bleeding. Diagn. Cytopathol. 1999;21:419-426.

This report describes a novel, reliable, and simplified approach for determination of intranuclear terminal deoxynucleotidyl transferase (TdT) expression. This approach utilizes standard permeabilization/fixation solutions to reliably detect intracellular antigens with minimal alterations in the light scatter properties of the analyzed cells. In contrast to other described methods, fluorescein isothiocyanate-conjugated anti-TdT antibody is added to previously analyzed and permeabilized cells after a leukemic cell population has been identified using characteristic surface staining patterns. The method eliminates the need for additional sample preparation or cumbersome permeablization steps and can easily be incorporated into any clinical laboratory's existing flow cytometry panels. Sixty-eight cases were analyzed with this method, including 31 acute myelogenous leukemias, 30 acute lymphoblastic leukemias, and 7 chronic lymphoproliferative disorders. To confirm the validity of the method, parallel immunoperoxidase staining and microscopic evaluation of cytocentrifuge test sample preparations were performed. Statistical analysis of the results reveals the method to be highly sensitive and specific, demonstrating exact correlation to the microscopic method. The ease and expeditiousness of this new procedure allows TdT testing to be routinely incorporated into the immunophenotyping repertoire of a busy clinical laboratory. In addition, the method should be readily adaptable to analyze a variety of other clinically relevant intranuclear and intracytoplasmic antigens.

Until now, commercially available syringe holders for fine-needle aspiration (FNA) were designed to be held in a pistol-grip manner. A newly developed, pencil-grip syringe holder, the Tao Aspirator™, was tested. The device is equipped with a release button for automatically drawing back the syringe plunger and a regulating knob for adjusting negative pressure for the aspiration. After direct smears were made for on-site examination, the remaining aspirated material was collected by rinsing the needle and syringe with CytoRich red fixative. Hettich cytocentrifuge preparations were then prepared. The quality of the first 150 FNA specimens procured by this device and prepared with liquid fixation was evaluated in terms of adequacy of specimen, amount of obscuring blood, preservation of cells, and ease of screening and interpretation. These 150 specimens included 32 from thyroids; 34 from breasts; 40 from lymph nodes; 24 from subcutaneous nodules; and 20 from salivary glands. There were no unsatisfactory specimens. In Hettich preparations, red blood cells were lysed, making interpretation easier. All cellular elements and tissue fragments were adequately fixed, showing excellent cellular morphology. Specimens fixed in liquid fixative yielded uniform cell suspensions, resulting in cytocentrifuge preparations with evenly distributed cells, and so the screening was also easier. The aspiration techniques using pistol-grip and pencil-grip FNA syringe holders were also compared in terms of control in tissue sampling, ease of use, and safety. The pencil-grip syringe holder allowed greater tactile sensation of the texture of the lesion, and enabled the operator to use a single hand to place a needle into a target lesion with minimal error. This device placed the hand relatively close to the needle tip while the hand was in a position of natural function, imparting more control in tissue sampling. It was more easily manipulated, and could prevent dripping when cystic fluid was aspirated. Specimen collection using the Tao Aspirator™ and processing with liquid fixation in addition to direct smear preparations allowed the laboratory to consistently produce adequate cytologic preparations and cell blocks. Diagn. Cytopathol. 1999;20:99–104. © 1999 Wiley-Liss, Inc.

The introduction of the laser scanning cytometer offers new capabilities in cell proliferation research, through its capacity for validation of each and every cell event through direct visualization on the microscope slide. In this study, we report a direct comparison of proliferation data derived from flow and laser scanning cytometry of human tumor nuclei labeled in vivo with bromodeoxyuridine (BrdUrd). Nuclear suspensions from 19 invasive ductal breast carcinomas and 12 gastric adenocarcinomas were prepared and analysed for BrdUrd uptake and DNA content. Specimens were analysed using a FACScan and then prepared on cytocentrifuge preparations for laser scanning cytometry. DNA index, labeling index (LI), duration of S-phase (Ts) and potential doubling time (Tpot) were calculated using standard procedures. There was an excellent correlation between the two techniques in the calculation of DNA index (R = 0.983, P > 0.0001) and LI (R = 0.924, P > 0.0001). The Ts proved more problematical (R = 0.448, P = 0.0115) but the Tpot showed closer agreement (R = 0.851, P > 0.0001) as the LI was the dominant determinant of Tpot. No single parameter could be identified as the major source of variation between the two techniques. We conclude that the laser scanning cytometer produces data equivalent to that obtained by flow cytometry.