Cytospin Centrifuge Research Articles

Enhanced visualization of articular cartilage chondroprogenitor-derived extracellular vesicles using cytospin centrifugation for confocal imaging

Characterizing Extracellular Vesicles (EVs) poses a challenge due to their complex composition and small size. Efforts to visualize fluorescently labeled EVs post-isolation with confocal microscopy encounter a significant obstacle in congregating the isolates on slides. While cytospin is a well-established method for cells, its application with EVs remains unexplored. This study aimed to isolate EVs and evaluate the efficiency of cytospin preparation to enhance their visualization using confocal microscopy. Following informed consent, cartilage shavings from a non-diseased human tibiofemoral joint were digested to obtain cartilage-resident chondroprogenitors through a migratory-assay (MCP). The MCPs were characterized for MSC markers (positive: CD105, CD73, CD90; negative: HLA-DR, CD34, CD45) using FACS. The conditioned medium was utilized to isolate EVs using the cutoff filter and precipitation technique, and their confirmation for transmembrane markers CD63 and CD81 was performed via FACS. Subsequently, the isolated EVs were subjected to confocal microscopy with and without (control) cytospin preparation. The MCPs exhibited high expression of positive and low expression of negative MSC markers. The study effectively employed cytospin centrifugation and confocal imaging to robustly identify EVs isolated from MCPs. Fluorescently labeled with CD63 and CD81 markers, the EVs isolated using cytospin centrifugation were concentrated on glass slides, and compared to control slides; the experimental setup facilitated a 100 times concentration of EVs, thereby enabling easy identification. Confocal analysis confirmed positive expressions of CD63 and CD81, aligning with FACS results. This study introduces a novel approach that enhances the visualization of MCP-derived EVs using cytospin centrifugation.

COMPARATIVE STUDY OF PLEURAL FLUIDS USING CYTOSPIN AND CONVENTIONAL CENTRIFUGE TECHNIQUE

To conduct a comparative study of pleural uids using Cytospin and conventional Centrifuge technique with emphasis on improvement of diagnosis yield in Tuberculosis effusions.

Role of Cytocentrifugation Combined with Nuclear Fast Picroindigocarmine Staining in Detecting Cryptozoospermia in Men Diagnosed with Azoospermia.

PurposeAzoospermia is defined as the absence of spermatozoa in the pellet of a centrifuged semen sample. In fact, when a basic semen analysis fails to detect sperm in the ejaculate, there is still the possibility of detecting rare sperm after centrifugation of the sample and examination of the pellet. In this study, we assessed the role of Cytospin centrifugation in combination with the nuclear fast picroindigocarmine (NF-PIC) staining in identifying sperm in azoospermic samples.Materials and MethodsSemen samples of 251 men diagnosed as having azoospermia after standard examination were further analyzed by Cytospin centrifugation in combination with NF-PIC staining.ResultsSperm were detected in 60 men (23.9%), thus changing their diagnosis to cryptozoospermia.ConclusionsBy identifying sperm in the semen of men who were thought to have total azoospermia, the Cytospin NF-PIC test can alter the diagnosis and further treatment of these men.

Clinical Profile of Bacterial Meningitis in Children and Comparative Inter-Alia Analysis of Various Microbiological Tests

AbstractAcute bacterial meningitis (ABM) is a life-threatening and neurologically debilitating infectious disease. We studied the clinical profile, organisms involved in bacterial meningitis in children, and compared the tests on cerebrospinal fluid (CSF), latex agglutination test (LAT), polymerase chain reaction (PCR), Gram stain (conventional) and Cyto-Tek cytospin centrifuge Gram stain to culture which is the gold standard. This was an observational cross-sectional study (age range 3 to 12 months) conducted in a tertiary care hospital, New Delhi, India over 1 year. A total of 101 patients were enrolled and divided into three age groups, namely, < 1 year, 1 to 5 years, and > 5 years. Fever was the most common presenting symptom in all groups (84.2%). Refusal to feed, headache, altered sensorium, vomiting, and blurring of vision were significantly associated with bacterial meningitis in all age groups. Cranial nerve palsies and neck rigidity were significantly higher in older children. Age < 5years, low-socioeconomic status, overcrowding, and smoke exposure were identified as risk factors for meningitis. Eight children died within 48 hours of admission and the rest (n = 93) recovered without complications. CSF culture was positive in 35.6% cases, with streptococcus pneumoniae being the most common organism. PCR was most sensitive (86.1%) and cytospin gram stain showed positivity in 65% cases which was statistically higher compared with conventional gram stain. Cytospin-prepared Gram stain was a viable low-cost alternative for early diagnosis of meningitis in low-income countries like India.

Enhanced microscopic diagnosis of oesophageal candidiasis through additional cytospin analysis of the fixative of oesophageal biopsies

To confirm or to refute the diagnosis of candida oesophagitis as the most common infectious disease of the oesophagus is a standard diagnostic procedure in histopathology. The fungal hyphae colonise mainly the superficial layers of the oesophageal squamous mucosa. Tangentially cut sections of oesophageal biopsies in the paraffin block might lead to a false negative result concerning mycotic infection. The aim of this study was to investigate whether cytospin analysis of the formalin fixative in which the biopsies were stored and transported would be a tool to close the diagnostic gap.Oesophageal biopsies from 150 consecutive patients with the clinical diagnosis or question "candida" or "candida oesophagitis" have been investigated. The biopsies were routinely processed and stained with haematoxylin and eosin and periodic acid-Schiff reaction. In parallel, the fixative fluid, usually disposed of after use, was processed by using a cytospin centrifuge and prepared for cytological proof of fungal hyphae. The cytology slides were also stained with periodic acid-Schiff reaction. In this blind study, the pathologist investigating the results of one procedure was unaware of the results of the second procedure.Out of 89 positive cytology cases, 64 cases (71,9 %) also showed a positive histology result. In the remaining 25 cases (28,1 %), fungal hyphae were seen only after re-evaluation of the original histology slides (n = 6) or in further serial sections using the complete tissue in the block (n = 5). In 14 cases, no hyphae could be detected histologically. Only in one of the 61 cytospin-negative cases was candida seen in histology.Our results show that diagnosing oesophageal candidiasis can be improved by more than one quarter using the formalin fixative for cytospin cytology.

Young Investigator Challenge: A novel, simple method for cell block preparation, implementation, and use over 2 years.

The cell block is an essential adjunct to conventional cytopreparatory techniques. The need for molecular analysis and immunostains will increase the need for successful cell block preparation. Even with this need, to the authors' knowledge very little has changed regarding the way in which cell blocks are produced. The authors developed A Formalin-Fixed, paraffin Embedded Cytology cell block Technique (AFFECT) that uses a cytospin centrifuge and funnel to deposit a cell pellet into a well on a piece of open-cell, absorbent foam. The foam and the pellet are then sent through normal processing. Herein, the authors present the implementation of this method and some of their experience with its performance over the course of 2 years. Although a comparison of the methods indicated good correlation for the production of a cell block between AFFECT and the agarose method, the AFFECT blocks demonstrated markedly improved cellular morphology. Over the first 6 months of use, AFFECT produced a successful cell block in 74% of cases overall, and in 65% of cases with a cell pellet measuring ≤0.1 mL. The year preceding the implementation of AFFECT and its first year of use were compared for endoscopic and bronchoscopic ultrasound-guided fine-needle aspiration specimens, and demonstrated an improved success rate. The authors developed a novel method of cell block preparation that demonstrates improved histology and has increased the success rate of cell block production compared with the agarose method. Cancer Cytopathol 2016;124:885-892. © 2016 American Cancer Society.

Sensitive detection of EML4-ALK fusion oncoprotein of lung cancer by in situ proximity ligation assay

EML4-ALK fusion oncogene has emerged as a novel molecular target in non-small cell lung cancer (NSCLC). Although break-apart fluorescent in situ hybridization (FISH) is the standard method for diagnosis, it is expensive, not readily available and sometimes difficult to interpret. In addition, ALK immunohistochemistry (IHC) may miss the diagnosis because of relatively low level of ALK transcription. In situ proximity ligation assay (PLA) originally developed for precise detection and quantification of proteins by dual recognition and amplification process was used for sensitive detection of EML4-ALK fusion oncoprotein in NSCLC cell lines (ALK negative cell: PC-9 and H460, ALK positive cell: H3122 and H2228). EML4-ALK oncogene and protein in lung cancer cells were confirmed by multiplex RT-PCR and Western blots. We detected 117 kDa variant 1 of EML4-ALK in H3122 and 90 kDa variant 3 of EML4-ALK in H2228. These cells were more sensitive to crizotinib, an ALK inhibitor compared with PC-9 and H460 cells without EML4-ALK rearrangement. After fixing on glass slides by cytospin centrifuge, in situ PLA test was performed. Among four cell lines, distinct, tiny spots were visible only in H3122 and H2228 cell lines with ALK rearrangement. The same results were also obtained when paraffin-embedded cell blocks were used. Highly specific and sensitive detection of EML4-ALK fusion oncoprotein is possible by in situ PLA method suggesting its clinical application.

Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs)

Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs), since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from antigen in surrounding non-glial cells. Yet, PAPs frequently display antigens related to motility and glia-synaptic interaction. Here, we describe the dissociation of morphologically intact glial cells (DIMIGs), permitting unambiguous antigen localization using epifluorescence microscopy. Astrocytes are dissociated from juvenile (p13–15) mouse cortex by applying papain treatment and cytospin centrifugation to attach the cells to a slide. The cells and their complete processes including the PAPs is thus projected in 2D. The entire procedure takes 2.5–3 h. We show by morphometry that the diameter of DIMIGs, including the PAPs is similar to that of astrocytes in situ. In contrast to cell culture, results derived from this procedure allow for direct conclusions relating to (1) the presence of an antigen in cortical astrocytes, (2) subcellular antigen distribution, in particular when localized in the PAPs. The detailed resolution is shown in an exemplary study of the organization of the astrocytic cytoskeleton components actin, ezrin, tubulin, and GFAP. The distribution of connexin 43 in relation to a single astrocyte's process tree is also investigated.

T(11;14)(q13;q32) in Mantle Cell Lymphoma Patients Is Present Only on Mature Malignant Cells. Fluorescence-Activated Cell Sorting and Fluorescence in Situ Hybridization Analysis

Abstract 4405 Background:Mantle cell lymphoma (MCL) is a distinct incurable B-cell neoplasm with a median survival of 3 to 5 years. t(11;14)(q13;q32) is the hallmark of the disease leading to overexpression of protooncogene cyclin D1. At least 60% of MCL patients have unmutated VH genes. Chemoresistance to conventional treatment and continuous disease relapses even after high dose therapy and autologous stem cell transplantation define the clinical course of the disease. A possible cause of this frequent failures may be that t(11;14)(q13;q32) occurs in early hematopoietic precursors, capable of multilineage differentiation. Aim:evaluation of t(11;14)(q13;q32) in different hematopoietic lineages of MCL patients. Methods:Bone marrow mononuclears from 12 MCL patients (including pleomorphic and blastoid variants) were sorted by Fluorescence-Activated Cell Sorting (BD FACSVantage SE) to divide the following cell lineages: CD45+CD34+ (progenitor cells); CD45+CD5+CD19+light chain Ig (mantle cell lymphoma); CD45+CD5-CD19+ (normal B-cells); CD45+CD14+ (monocytes); CD45+CD3+ (T-cells); CD45-GlyA+ (erythrokaryocytes) and granulocytes by light scattering, excluding CD14+CD45+ cells. The purity of sorted cells was checked by flow cytometry (BD FACSCanto II) and correlated to the number of sorted cells – if there were more than 50 thousands cells the purity was more than 92% (usually more 97%), but if there were less than 20 thousands cells the purity was 80% (never less). After sorting cells in the test-tube were washed from the PBS, fixed in methanol and glacial acetic acid mixture and layered onto slides by Cytospin centrifuge (Cellspin II Tharmac). The probes have been denatured at 75°C for 2 minutes, and hybridized with dual fusion LSI IGH-CCND1 probes (Vysis Inc.) at 37°C for 17–20 hours. Signals were visualized with Olympus IX-61 microscope with triple filter cube (DAPI / FITC / TexasRed). Results:t(11;14)(q13;q32) was present in 97% of sorted mantle cell lymphoma cells (range 84–100%), whereas in cells of all other lineages including normal B-cells t(11;14)(q13;q32) was not found at all. However, direct assessment of t(11;14)(q13;q32) in CD34+CD45+ cells and in normal B-cells was difficult due to the small number of these cells. In one case (classic variant MCL) we have sorted two “independent” cell populations 1 log different in the level of expression CD5: CD5dimCD19+ and CD5+CD19+. In both subpopulations t(11;14)(q13;32) was detected in 98,5% cell nuclei, indicating no correlation between CD5 level and fusion gene expression. In three cases using together FACS and FISH we were able to detect the population of MCL cells in bone marrow as small as 0,1–0,05%, whereas histology examination and standard FISH analysis failed to detect bone marrow involvement. Conclusion:Out data give evidence for the absence of t(11;14)(q13;q32) in early hematopoetic cells. FACS allows increase FISH sensitivity in 100–1000 times. Disclosures:No relevant conflicts of interest to declare.

Computerized cell-scanning system for evaluating human spermatogenesis in non-obstructive azoospermic patients

There may be incompatibility between testicular histopathological evaluation and testicular sperm extraction (TESE) outcome. Assessment for sperm presence and different pathological disturbances of non-obstructive azoospermia (NOA) remains challenging. An assay for maximal sampling and accurate identification of testicular cells from NOA patients undergoing TESE and autopsied fertile controls was developed. Testicular cells stained and scanned automatically for morphology underwent fluorescence in-situ hybridization using centromeric probes for chromosomes X, Y and 18 after destaining. Cells were automatically classified according to ploidy, and ratios of haploid cells and autosomal (18) and sex-chromosome bivalent rates were calculated. Identification of testicular cells in suspension enabled prediction of spermatogenesis in seven of eight Sertoli-cell-only syndrome patients. Haploid/diploid cell ratios were 67.6:32.2 for controls and 9.6:90.4 for patients. Both autosomal (18) and sex-chromosome bivalents were present in patients (4.1 ± 5.82%) and controls (19.7 ± 8.95%). Few tetraploid pachytene spermatocytes were observed. More secondary spermatocytes with NOA showed two distinct signals for chromosome 18 (27.9 ± 32.69%) compared with controls (0.4 ± 0.35%). The computerized cell-scanning system enables simultaneous application of morphology and chromosome analysis of testicular cells, which enhance assessing different pathological disturbances and estimating the likelihood of a successful second TESE procedure. Assessment for sperm presence and different pathological disturbances of non-obstructive azoospermia (NOA) remains challenging. There may be incompatibility between testicular histopathological evaluation and testicular sperm extraction outcome. Precise identification of individual germ cells in a wet cell preparation during TESE could contribute to understanding the pathology. An assay for maximal sampling and accurate identification of testicular cells from NOA patients undergoing TESE was developed. A prospective cohort study was conducted at a university-based IVF centre. Cells from the testicular suspension of 12 NOA patients and three autopsied fertile controls were fixed on glass slides using a Cytospin centrifuge and stained with May–Grünwald Giemsa. The slides were automatically scanned for cell morphology. Fluorescence in-situ hybridization using centromeric probes for chromosomes X, Y and 18 was performed after destaining, and cells were automatically classified according to ploidy. Identification of different testicular cells in the suspension enabled prediction of spermatogenesis in seven out of eight Sertoli-cell-only syndrome patients. Ratios of haploid to diploid cells were 67.6:32.2 in the controls and 9.6:90.4 in the study group. Presence of both autosomal (18) and sex chromosome bivalents was 19.7 ± 8.95% in the controls and 4.1 ± 5.82% in the study group ( P < 0.05). Some tetraploid pachytene spermatocytes were identified in both groups. More secondary spermatocytes in the study group showed two distinct signals for chromosome 18 than in controls: 27.9 ± 32.69% versus 0.4 ± 0.35 ( P < 0.02). Thus, computerized cell scanning enables simultaneous application of morphology and chromosomal analysis of testicular cells and examination of meiotic pairing for enhanced assessment of pathological disturbances.

Somatic (CSS) and differential cell count (DCC) during a lactation period in ass’milk

Hypoallergenic properties of ass’s milk protein fractions have been recently confirmed, allowing ass’s milk to be considered as a valid substitute of the available hypoallergenic infant formulas. The objective of this study was to give a further contribution to the knowledge of ass’s milk safety and quality characteristics. A new procedure has been developed with a cytospin centrifuge in differential counts of milk somatic cells. Somatic cells count (SCC), differential somatic cells count (DCC) and cultural examinations have been carried out in 62 milk samples collected from 11 asses at three different stages of lactation. Four major cells populations had been identified in ass’s milk too: lymphocytes (Ly), monocytes/macrophages (MA), polymorphonuclear neutrophils (PMNL), and epithelial cells (CE). The patterns of these cells have been discussed in comparison with cells found in dairy cows and ewes milk. In conclusion, a reproducible standard procedure has been developed to determine cell count of ass’s milk.

Down-regulation of Caveolin-1, an Inhibitor of Transforming Growth Factor-β Signaling, in Acute Allergen-induced Airway Remodeling

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-beta (TGF-beta). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-beta, perhaps by forming membrane invaginations that enfold TGF-beta receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-beta signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-beta signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-beta signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-beta and thereby ameliorating pathological airway remodeling.

Expression of Sialidase Neu2 in Leukemic K562 Cells Induces Apoptosis by Impairing Bcr-Abl/Src Kinases Signaling

Chronic myeloid leukemia is a hematopoietic stem cell cancer, originated by the perpetually "switched on" activity of the tyrosine kinase Bcr-Abl, leading to uncontrolled proliferation and insensitivity to apoptotic stimuli. The genetic phenotype of myeloid leukemic K562 cells includes the suppression of cytosolic sialidase Neu2. Neu2 transfection in K562 cells induced a marked decrease (-30% and -80%) of the mRNA of the anti-apoptotic factors Bcl-XL and Bcl-2, respectively, and an almost total disappearance of Bcl-2 protein. In addition, gene expression and activity of Bcr-Abl underwent a 35% diminution, together with a marked decrease of Bcr-Abl-dependent Src and Lyn kinase activity. Thus, the antiapoptotic axis Bcr-Abl, Src, and Lyn, which stimulates the formation of Bcl-XL and Bcl-2, was remarkably weakened. The ultimate consequences of these modifications were an increased susceptibility to apoptosis of K562 cells and a marked reduction of their proliferation rate. The molecular link between Neu2 activity and Bcr-Abl signaling pathway may rely on the desialylation of some cytosolic glycoproteins. In fact, three cytosolic glycoproteins, in the range 45-66 kDa, showed a 50-70% decrease of their sialic acid content upon Neu2 expression, supporting their possible role as modulators of the Bcr-Abl complex.

Quantitative Proteomic Analysis Using Isobaric Protein Tags Enables Rapid Comparison of Changes in Transcript and Protein Levels in Transformed Cells

Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.

Immune cells in the anterior chamber of patients with immune reactions after penetrating keratoplasty.

Lymphocytes, monocytes, and macrophages are the predominating immune cells in graft rejection after keratoplasty in animal models. This study focuses on the isolation of immune cells from the anterior chamber of patients with slight, moderate, and severe endothelial immune reactions after penetrating keratoplasty. Anterior chamber puncture was performed in five patients with cataract without inflammation and without penetrating keratoplasty (C1), in three patients undergoing penetrating keratoplasty without immune reactions (C2), in four patients undergoing penetrating keratoplasty after complete resolution of endothelial immune reactions (C3), in seven patients undergoing penetrating keratoplasty with slight endothelial immune reactions (IMI), in 10 patients undergoing penetrating keratoplasty with moderate endothelial immune reactions (IM2), and in eight patients undergoing penetrating keratoplasty with severe endothelial immune reactions (IM3). In each patient, approximately 0.1 mL of aqueous humor was examined. Cells in suspension were directly centrifuged on glass slides using a Cytospin centrifuge, stained, and evaluated under the light microscope. Groups C1, C2, and C3 did not contain cells. Immune cells were identified in three of seven patients in IM1, in eight of 10 patients in IM2, and in eight of eight patients in IM3. Predominating cells were macrophages and monocytes followed by lymphocytes. Regarding all patients in IMI, IM2, and IM3, a statistically significant correlation between detected cells and patient age, period between penetrating keratoplasty and anterior chamber puncture, or period between first symptoms and anterior chamber puncture could not be revealed. Granulocytes were found statistically significantly less often in patients with high-risk indications, in patients with a history of immune reactions and under immunosuppression. Lymphocytes were found statistically significantly less often in patients with a history of immune reactions. The probability to isolate immune cells from the anterior chamber of patients undergoing penetrating keratoplasty correlates with the severity of the endothelial immune reactions. This study is a first step to evaluate how detailed immunologic findings from animal keratoplasty models fit to clinical reality in patients undergoing keratoplasty. In the next step, cells found in the aqueous humor of patients with endothelial immune reactions should be further characterized directly (determination of molecules on the surface of the cells) or indirectly (determination of cytokine levels in the aqueous humor).

Prognostic significance of free gastrointestinal tumor cells in peritoneal lavage detected by immunocytochemistry and polymerase chain reaction.

The aim of our study was to identify tumor cells in peritoneal lavage comparatively with immunocytochemistry (ICC) and half-nested reverse transcriptase-polymerase chain reaction (RT-PCR) using carcinoembryonic antigen (CEA) as marker and to evaluate their prognostic significance. In 75 patients who underwent surgery for a carcinoma of the colorectum (n=49), stomach (n=17) or pancreas (n=9) and 13 patients with an abdominal aortic aneurysm (control group) the abdomen was irrigated with saline solution immediately after laparotomy. Cells were separated by Ficoll-density centrifugation and divided into 2 equal volumes for ICC and RT-PCR. For ICC cells were spun onto slides by cytospin centrifugation and stained with a monoclonal antibody (mab) against CEA using the APAAP method. For RT-PCR total RNA was extracted from the cells, transcribed into cDNA and amplified with CEA-specific primers. Lavages of 13 patients with an abdominal aortic aneurysm and blood samples of 6 healthy donors served as controls. Immunostained tumor cells were found in peritoneal lavage in 23% (17/75) of all patients, whereas 63% (47/75) of patients gave a positive result by RT-PCR analysis. In the control group (n=13) no patient presented with tumor cells in ICC, however 5 of 13 (38%) showed amplified CEA-mRNA by RT-PCR, and so did one of six blood samples. Using ICC technique, we found significant correlations between detection rates and pT-, pN-, pM-categories as well as tumor stage. On the contrary, by RT-PCR significant correlations were observed only between pT- and pM-categories and detection rates. Detection of tumor cells in peritoneal lavage with both techniques was associated with poor prognosis. Moreover, these tumor cells are an independent prognostic factor and may have an influence on the development of peritoneal carcinomatosis. ICC is a useful method for detection of tumor cells in peritoneal lavage. In contrast, half-nested RT-PCR cannot be recommended, as the detection rates are unproportionally high, obviously as a result of CEA-mRNA expression in nontumor cells.

Cytokine-induced nitric oxide production of joint cartilage cells in continuous passive movement. Anti-inflammatory effect of continuous passive movement on chondrocytes: in vitro study

The temporomandibular joint is a place of motion where release of proinflammatory cytokines like interleukin-1 beta (IL-1 beta) induces cartilage destruction via production of nitric oxide (NO). The purpose of this study was to evaluate the effects of continuous passive motion in the form of cyclic stretch on the synthesis of inducible nitric oxide synthase (iNOS). Articular chondrocytes were harvested from rabbit cartilage slices and cultured in F12 medium supplemented with 10% fetal calf serum. Subsequently, cells (10(5)/ml per well) were transferred to Flexercell plates, grown to 90% confluency, and subjected to one of the following regimens: (1) no mechanical strain (10 Hz, 20% elongation rate); (2) rhIL-1 beta (1 ng/ml); (3) mechanical strain; (4) mechanical strain and rhIL-1 beta. The cells were exposed to cyclic stretch for 24 h. Thereafter, cells were trypsinized and centrifuged on microscope slides in a cytospin centrifuge. The presence of iNOS was determined by indirect immunoperoxidase staining using polyclonal rabbit anti-iNOS as primary antibodies, and goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) as secondary antibodies. Diaminobenzidine was used as substrate for HRP. Total NO production was spectrophotometrically measured in the supernatants of cultures using Greiss reaction. All experiments were done in triplicates and the statistical significance was analyzed by Student's t-test. Cells with treatment 1 and 3 did not exhibit presence of iNOS. IL-1 beta-treated cells in group 2 exhibited intense peroxidase staining. Cells in group 4 exposed to IL-1 and mechanical stress showed staining with considerably less intensity. Control cells treated with normal rabbit IgG as a primary antibody did not exhibit peroxidase staining. The data were further confirmed by measurement of statistically significant differences of NO levels in supernatants assessed in the four different treatment groups. Our findings suggest that continuous passive motion exerts anti-inflammatory effects on chondrocytes, downregulating the quantity of iNOS-positive cells. Since NO acts as an intracellular, transcellular, and cytotoxic molecule, it has a basic importance for proper post-traumatic and postoperative TMJ function as well as the course and therapy of inflammatory TMJ diseases.

In Vivo Genotoxicity of Crystalline Silica as Evidenced by Micronuclei in Pulmonary Alveolar Macrophages: Low-Dose Study

We have previously published results showing time-course data (Leigh et at., 1998) and a dose-response relationship between macrophage micronucleus formation and crystalline silica dose in intratracheally instilled rats at 2.5, 7.5, and 22.5 mg dosage, without an inert dust control (Wang et al., 1997). We here extend this study to low dose (0.025, 0.25, and 2.5 mg crystalline silica) with 2.5 mg TiO2 control. Specific-pathogen-free male Wistar rats were intratracheally instilled with 0.5 ml saline, and 0.025 mg, 0.25 mg, or 2.5 mg crystalline silica (Min-U-Sil 5) and 2.5 mg TiO2 suspended in 0.5 ml saline (5 rats in each group). Five days after instillation, rats were sacrificed and 10 ml of bronchial alveolar lavage fluid was obtained. A 100-μ1 volume was placed on slides by Cytospin centrifugation, stained with Diff-Quik, and 1000 macrophages were scored for micronuclei (defined by diameter < half main nucleus; same staining; round shape and complete separation). Micronucleus incidence was significantly elevated (p < .01) at the lowest crystalline silica dose compared with saline control. There was a dose-response relationship with crystalline silica exposure. Numbers (mean ± SEM) of micronucleated macrophages per 1000 macrophages scored were 1.5 ± 0.5 (saline), 3.3 ± 0.3 (0.025 mg crystalline silica), 7.1 ± 0.4 (0.25 mg crystalline silica), 10.1 ± 0.3 (2.5 mg crystalline silica), and 0.9 ± 0.3 (2.5 mg TiO2). We conclude that intratracheal instillation of low doses of crystalline silica can induce micronucleus formation in alveolar macrophages in a dose-related manner. We further believe that this is not a nonspecific effect, consistent with crystalline silica being a genotoxic carcinogen.

Prenatal genetic diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative and positive magnetic activated cell sorting.

Fetal nucleated red blood cells (nRBCs) are rare in maternal circulation, but their presence constitutes a potential source of non-invasive prenatal genetic diagnosis. This study was undertaken to establish a non-invasive prenatal genetic diagnosis method using isolated fetal nRBCs. A multi-step method including triple density gradient and magnetic activated cell sorting (MACS) using CD45 and CD71, cytospin centrifugation, K-B staining, and glycophorin A-immuno fluorescence in situ hybridization (GPA-immuno FISH) was performed. The study population included 65 patients from 8 to 41 weeks of gestation, and fetal nRBC was separated from all cases. The number of fetal nRBCs retrieved was 12.8 +/- 2.7 in 8 to 11 gestational weeks, 15.2 +/- 6.5 in 12 to 18 gestational weeks, 16.4 +/- 6.5 in 19 to 23 gestational weeks, 10.6 +/- 3.2 in 24 to 28 gestational weeks, and 5.5 +/- 1.9 in 35 to 41 gestational weeks: the mean number of nRBCs collected from 20 ml of maternal peripheral blood was 13.7 +/- 6.2. The highest value of yield was 45.6% from 12 to 18 weeks gestation. The fetal sex determination confirmed by amniocentesis or chorionic villus sampling showed 100% sensitivity and 91.7% specificity for males; 91.7% sensitivity and 100% specificity for females. We showed that fetal cells can be reliably enriched from maternal blood and that they can be used for detecting specific chromosomes by FISH with a specificity superior to current non-invasive methods.

Prevalence of High Somatic Cell Counts in Bulk Tank Goat Milk

Higher SCC have been observed in goat milk than in cow milk. This study evaluated the prevalence of high SCC and determined whether increased neutrophils were a factor in high SCC. Bulk tank goat milk samples were collected from commercial dairy goat herds in California, Arkansas, Michigan, and Wisconsin. Total SCC were determined using a Fossomatic Electronic Cell Counter and microscopically using pyronin Y-methyl green stain. Differential counts were determined using Wright's-stained cytospin centrifuge preparations. Average SCC were high (6.12 log10 cells/ml; geometric mean, 1.32×106 cells/ml). The average of the Fossomatic and direct microscopic SCC indicated that 8.6% of the producers had SCC <.75×106 cells/ml and that 34.5% were <1×l06 cells/ml. The current goat milk SCC standard is 1×106 cells/ml; the cow milk SCC standard will be .75×106 cells/ml in 1993. Milk samples contained 9.9% macrophages, 2.8% lymphocytes, and 87% neutrophils. These data indicate that high SCC were prevalent and that increased neutrophils contributed to high SCC in the bulk tank goat milk.