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PubMed1993

Phase contrast microscopic examination of urinary erythrocytes to localise source of bleeding: an overlooked technique?

AIMS: To localise the source of bleeding in the urinary tract in patients presenting with haematuria. METHODS: Urine samples were obtained from 109 patients with symptoms referable to the urinary tract. The sample was examined for the presence of red blood cells by phase contrast microscopy (PCM) and the proportion of dysmorphic and isomorphic red blood cells was determined. If more than 20% of the red blood cells were dysmorphic a glomerular origin for the site of bleeding was suspected; if less than 20% of the red blood cells were isomorphic a non-glomerular origin was suspected. Phase contrast microscopy and clinical findings were correlated. RESULTS: The correct bleeding site was shown in 27 of 30 (90%) patients with glomerulopathy and in all 17 patients with bleeding from the lower urinary tract, indicating that this method of analysis has a sensitivity of 90% and specificity of 100% for detecting the glomerular source of bleeding. CONCLUSIONS: The examination of urine for dysmorphic and isomorphic red blood cells by phase contrast microscopy is strongly recommended in routine clinical practice for the detection of glomerular and non-glomerular lesions. This technique may avoid unnecessary investigations for the diagnosis of the site of bleeding in patients with haematuria.

Adult↗
PubMed1957

An analysis of myogenesis by the use of fluorescent antimyosin.

Antibodies against myosin of adult chicken skeletal muscle were labelled with fluorescein and used as staining reagents to analyze the development of trunk myoblasts in the chick embryo. Myoblasts from the brachial myotomes were studied in three ways: (a) Specimens were fixed, sectioned, and stained with iron-hematoxylin. (b) Living myoblasts, and myoblasts prepared by glycerol extraction, were teased and examined by phase contrast microscopy. (c) Embryo trunks were treated with fluorescent antimyosin or with a control solution of fluorescent normal globulin, and were examined by fluorescence and phase contrast microscopy. Both glycerol-extracted and fixed materials were used. Cross-striated myofibrils appeared first in stage 16 to 17 embryos in the series studied by antimyosin staining and fluorescence microscopy. Striated myofibrils appeared first in stage 18 to 19 embryos, in the series stained by iron-hematoxylin, and at stage 22 to 23, in the series studied by glycerol extraction and phase contrast microscopy. In each series, myofibrils without apparent cross-striations were detected shortly before cross-striations were observed. Specific staining by antimyosin occurred only in differentiating myoblasts. Within the myoblasts antimyosin staining was confined to the A bands of the slender myofibrils. The following observations suggest that the first delicate striated structure to appear in the early 3 day myoblast was remarkably mature: (1) The sarcomere pattern both in length and in internal detail, was similar to that of adult muscle. (2) The distribution of myosin, as revealed by antimyosin staining, was the same in the embryonic as in the mature myofibril. (3) Glycerol-extracted myoblasts contracted vigorously on exposure to ATP. The changes in sarcomere band pattern were indistinguishable from those occurring during contraction of adult muscle induced by ATP. (4) ATP contraction was blocked by prior antimyosin staining in embryonic myoblasts as in mature muscle. It is suggested that the early myofibril grows laterally as a thin sheet associated with the sarcolemma, and that growth in length occurs in the growth tips of the elongating myoblast.

Animals↗
PubMed2002

Capsule-substrate contact deformation: determination of adhesion energy.

A study is reported of a cellular entity (liquid-filled microcapsule) adhered on a flat glass substrate in response to changes in osmotic pressure and temperature. High-resolution reflection interference contrast microscopy (HR-RICM) and phase-contrast microscopy were developed for probing the adhesion contact area, capsule-substrate separation profile and adhesion energy of the adhering microcapsule. The new technique increased the detection limit of the measured capsule wall-substrate separation in the cohesive zone from 1 to 4.5 microm and improved the spatial resolution of the heterogeneous contact zones. A theoretical model was applied to correlate quantitatively the adhesion energy to the area of the contact zone. The work demonstrated the possibility of ascertaining the quantitative interfacial adhesion energy of a liquid-filled microcapsule using the present technique and represents the first step in extending this novel approach to study more complicated systems, such as cell-substrate interactions, in the future.

Animals↗
PubMed1984

Influence of cefmenoxime, ceftriaxone, latamoxef, and ceftazidime on the lysis of Klebsiella pneumoniae: a light and electron microscopic study.

The influence of antibiotics on the morphologic alterations of the cell wall of Klebsiella pneumoniae serotype K 17 has been studied by electron microscopy as well as by phase-contrast microscopy. Ceftazidime, cefmenoxime, ceftriaxone, and latamoxef were the antibiotics used. The effect of each concentration between 0.03 and 1,024 mg/liter was investigated on 5 X 10(7) bacteria per milliliter. Two different reactions could be detected: ceftazidime and latamoxef produced at first long forms and suddenly lysis; cefmenoxime and ceftriaxone produced at first long forms, later spherical forms, and then gradually lysis.

Bacteriolysis↗
PubMed1981

Characterization of polysaccharide accumulation in a cell division defective mutant of Escherichia coli 15T-.

Escherichia coli 15T-R1, a temperature-dependent cell division mutant, grows into filaments of various lengths (200 to 500 microgram) at 24 degrees C, but divides essentially normally at 37 degrees C. When grown to late-exponential phase at the restrictive temperature, the elongated cells showed discrete areas of increased density at polar regions and other sites in the cytoplasm, when viewed by phase contrast microscopy. Electron microscopy of preparations specifically stained for polysaccharide revealed clusters of granules with a similar distribution pattern to that of the dense areas seen by phase contrast microscopy. The granules were susceptible to alpha-amylase digestion, and chemical analysis of the extracted and purified polysaccharide showed that it consisted of polyglucose, including glycogen. At 24 degrees C the R1 cells contained about twice as much polyglucose and four times as much glucogen as at 37 degrees C.

Cell Division↗
PubMed1986

Subgingival microflora in bleeding and nonbleeding pockets.

The subgingival flora of bleeding and nonbleeding 4-6 mm pockets was investigated using phase-contrast microscopy. Subgingival plaque was sampled from 11 patients with generalized moderate periodontitis. 4 subgingival samples were obtained from each patient, 2 from sites that bled upon standardized probing force and 2 from sites that did not. The amounts of gingival inflammation, supragingival plaque, attachment level and pocket depth were also assessed at each site. The %s of 4 bacterial morphotypes were assessed using phase-contrast microscopy. No significant differences were found in the %s of cocci, motile rods, or spirochetes between bleeding and nonbleeding sites. Significant correlations were found, however, between the % of spirochetes and probing depth, attachment level, and gingival inflammation. The observations indicate that the use of bleeding on probing may not be justified as an indicator of infection by those "periodontopathic" bacteria identifiable by phase-contrast microscopy. However, limitations in the microscopic method may have prevented us from observing differences between the 2 types of sites on a species level.

Adult↗
PubMed1985

Airborne asbestos in Colorado public schools.

Levels of airborne asbestos for six Colorado public school facilities with sprayed-on asbestos materials were documented using three analytical techniques. Phase contrast microscopy showed levels up to the thousandths of a fiber per cubic centimeter (f/cc), scanning electron microscopy (SEM) up to the hundredths of a f/cc, and transmission electron microscopy coupled to selected area electron diffraction and energy dispersive X-ray analysis (TEM-SAED-EDXA) up to the tenths of an asbestos f/cc. Phase contrast microscopy was found to be an inadequate analytical technique for documenting the levels of airborne asbestos fibers in the schools: only large fibers which were not embedded in the filter were counted, and asbestos fibers were not distinguished from nonasbestos.

Air Pollutants↗
PubMed1964

ASSEMBLY AND AGGREGATION OF TOBACCO MOSAIC VIRUS IN TOMATO LEAFLETS.

Cells of tomato leaflets (Lycopersicum esculentum Mill.) were studied by phase and electron microscopy at various intervals after inoculation with a common strain of tobacco mosaic virus (TMV). Forty-eight hours after inoculation, prior to the development of assayable virus, individual TMV particles, and also particle aggregates, were observed in the ground cytoplasm of mesophyll cells. The most rapid synthesis of virus occurred between 80 and 300 hours after inoculation. Cytological changes during this time were characterized by an increased number of individual particles in the cytoplasm, growth of some aggregates, distortion and vacuolation of chloroplasts, and formation of filaments in the cytoplasm which were approximately four times the size of TMV. These filaments were interpreted as possible developmental forms of the TMV particle. Vacuoles in chloroplasts commonly contained virus particles. Evidence indicated that TMV was assembled in the ground cytoplasm and, in some cases, subsequently was enveloped by distorted chloroplasts.

Chloroplasts↗
PubMed2004

The partial head decondensation test is a new, quick method to assess acrosome status in human spermatozoa.

OBJECTIVE: To develop a fast method for assessing acrosome status in human spermatozoa. DESIGN: Development of a new in vitro test to assess acrosome reaction in human spermatozoa. SETTING: Academic medical institution. PATIENT(S): Normozoospermic subjects. INTERVENTION(S): Spermatozoa were isolated from fresh semen samples, capacitated, and stimulated or not with P or ionomycin. Acrosome reactions were evaluated by phase-contrast microscopy after a brief sperm incubation in a decondensing solution. The results were compared with those obtained by scanning electron microscopy and fluoresceinated lectin staining. MAIN OUTCOME MEASURE(S): Percentage of intact acrosomes. RESULT(S): The new procedure allowed intact acrosomes to be easily identified and quantified by phase-contrast microscopy. In unstimulated and ionomycin-treated spermatozoa, a very good agreement was found among the new test, scanning electron microscopy, and fluoresceinated lectin staining. In P-treated spermatozoa, the proposed method allowed a significantly higher percentage of reacted acrosomes to be resolved, likely due to its ability to detect the very initial stages of the acrosome reaction. CONCLUSION(S): The new test allows acrosome-intact and acrosome-reacted spermatozoa to be unambiguously singled out and quantified. The method is rapid, reliable, sensitive, and easy to perform, which makes it of profitable use in both basic research and diagnostic practice.

Acrosome Reaction↗
PubMed1989

[Basic studies on CaO-P2O5-MgO-SiO2-CaF system glass ceramics. 1. Morphology under the phase-contrast microscope and growth of cultured cells].

In order to determine the biocompatibility of glass ceramics which is one of the new biomaterials, in vitro studies were carried out by a cell culture method using four established cell lines. Materials used were glass ceramic disks with a diameter of 3 mm, and polystyrene coverslips of the same size as controls of the growth curve. Cells of each line were inoculated into 24-well multiplates at an appropriate density onto glass ceramic disks, and examined by phase contrast microscopy on the 1st, 3rd, 6th and 8th day. In addition, doubling time and saturation density were calculated from the growth curve. The results obtained were as follows. 1) Phase-contrast microscopy revealed that cells of each line attached to the disk within 24 hours and their numbers increased with time. After 8 days of cultivation, all of them reached confluence. 2) Contact with the glass ceramics did not cause cellular death or degeneration. Furthermore, the cultured cells showed the same morphological features as the control cells. 3) According to the growth curves, doubling time of all cells cultured with glass ceramics was shorter than that of the control cultures. On the other hand, saturation density was reduced to a minimum of 80% of the controls. These findings led to the conclusion that glass ceramic materials do not prevent the growth of cultured cells. According to the above results, glass ceramics possess the characteristics needed for bone grafts and implant materials.

Cell Line↗
PubMed1978

Enzyme-linked immunosorbent assay for detection of hepatitis A antigen in stool and antibody to hepatitis A antigen in sera: comparison with solid-phase radioimmunoassay, immune electron microscopy, and immune adherence hemagglutination assay.

Previously described techniques for detection of hepatitis A antigen (HA Ag) and antibody (anti-HA) have required purified HA Ag and expensive equipment. Herein is described an enzyme-linked immunosorbent assay (ELISA) for specific detection of HA Ag in human stool filtrates and of anti-HA in sera by using selected HA Ag-containing human stool filtrates as the antigen source. Because human stools often react nonspecifically in serological tests for HA Ag, blocking with preexposure and hyperimmune anti-HA sera from a chimpanzee inoculated with hepatitis A virus was used to confirm specific detection of HA Ag. The sensitivity of ELISA was found to be comparable to that of solid-phase radioimmunoassay (SPRIA) and immune electron microscopy (IEM). Of 37 acute-phase stools collected from nine patients, 16 were positive for HA Ag by ELISA. In 13 of these, HA Ag particles were found by IEM, and an additional 3 stools negative by ELISA contained HA Ag particles by IEM. Eight control stools were negative by both ELISA and IEM. Anti-HA was measured in sera by demonstrating its ability to block binding of the enzyme conjugate to HA Ag in a stool without detectable nonspecificity. This test (blocking ELISA) was as sensitive and specific as blocking SPIRA, IEM, and immune adherence hemagglutination and, like SPRIA and IEM, detected early-developing antibody. The ELISA is simple to perform and requires only a minimum of equipment. It is useful for screening stools for HA Ag and for monitoring HA Ag during purification, as well as for detecting early and late anti-HA in sera.

Acute Disease↗
PubMed2005

"Decoy cells" in urine.

BK virus-associated nephropathy is an emerging cause of kidney transplant loss. Tapering immunosuppressive drugs and antiviral agents are the only therapy. The diagnosis is based on immunohistochemical findings or polymerase chain reaction on renal biopsy. Phase-contrast microscopy without staining is a simple test to screen the urine of transplant recipients for BK nephritis. Any kidney transplant unit should have the ability to detect decoy cells by phase-contrast microscopy on a spot urine.

BK Virus↗
PubMed1995

Validity of G1-cells in the differentiation between glomerular and non-glomerular haematuria in children.

Urine samples from 100 children and adolescents with micro- or macrohaematuria were investigated using phase contrast microscopy to establish the percentage of G1-cells that could differentiate glomerular from non-glomerular haematuria. The G1-cell is a special form of dysmorphic erythrocyte which seems to be specific for glomerular haematuria. Glomerular haematuria, defined by clinical criteria from biopsy, physical examination, standard laboratory evaluation and family history, was observed in 51 patients (group 1). Non-glomerular haematuria was found in 49 patients (group 2). The latter group had urinary tract infections, urolithiasis, hypercalciuria or haematuria caused by urological operation or diagnostic procedure. The percentage of dysmorphic erythrocytes differed significantly between the two groups studied (42 +/- 3% in group 1 vs. 6 +/- 1% in group 2, mean +/- SEM, P < 0.01); there was also a significant difference in G1-cells (19.4 +/- 1.7% in group 1 vs. 0.6 +/- 0.2% in group 2, mean +/- SEM, P < 0.01). When glomerular haematuria was defined on the basis of > or = 30% dysmorphic erythrocytes by phase contrast microscopy, sensitivity, specificity and efficiency were 71%, 100% and 85%, respectively. When glomerular haematuria was defined on the basis of > or = 5% G1-cells, sensitivity, specificity and efficiency were 100%, 100% and 100%, respectively. The differentiation of glomerular and non-glomerular haematuria in children by determination of G1-cells appears to be more sensitive and efficient than the determination of the percentage of dysmorphic erythrocytes by phase contrast microscopy.

Adolescent↗
PubMedDate not supplied

In vivo confocal microscopy in the acute phase of corneal inflammation.

Two cases of noninfectious keratitis were studied using in vivo corneal confocal microscopy in addition to routine slit-lamp biomicroscopy. A 10-year-old boy suffered from keratitis in his left eye due to a bee sting and a 20-year-old man had keratitis following corneal blunt trauma. In both cases, we found a honeycomb pattern at the anterior and mid-stromal level of the middle cornea. This honeycomb pattern disappeared in 1 week with steroid treatment. This pattern might be caused by syncytial cell bodies of activated keratocytes, by focal corneal stroma edema, or by polymorphonuclear leukocyte infiltration along with the keratocytes or along preformed channels within the corneal stroma known as Bowman channels. Further analysis in a large number of patients may aid in further understanding in vivo human corneal inflammation.

Acute Disease↗
PubMed1982

In vitro correlates of endothelial injury and repair.

The effects of immune complexes, microbial components, and glomerular basement membrane on cultured human umbilical endothelial cells were assessed in vitro using phase contrast microscopy, cell counts, 51Cr release, and terminal tritiated thymidine-labeling assays. Neither heat-aggregated IgG nor bovine serum albumin-antibovine serum albumin immune complexes altered endothelial cell growth as measured by cell counts, thymidine labeling, and phase contrast microscopy, nor did immune complexes induce cytotoxicity as measured by 51Cr release. Some microbial components appeared cytotoxic to endothelial cells, other microbial components had no effect, and one, clostridial neuraminidase, induced proliferation of endothelial cells as measured by phase contrast microscopy, cell counts, and thymidine labeling. Native and glycosidase-altered glomerular basement membrane also enhanced endothelial cell thymidine labeling. These studies show that microbial components directly alter endothelial cell growth in vitro, whereas immune complexes by themselves do not. Thus, in vivo microbial components by themselves may directly affect endothelial cells and cause vascular pathology, whereas immune complexes presumably must recruit other inflammatory systems to affect endothelial cells. Alterations of basement membrane during inflammation may also affect endothelial growth and, thus, vascular pathology.

Antigen-Antibody Complex↗
PubMed2001

[European-American-type hairy cell leukemia without splenomegaly, treated successfully with deoxycoformycin].

A 55-year-old Japanese man was hospitalized on October 5, 1999, because of high fever. Physical examination revealed neither lymphadenopathy nor hepato-splenomegaly. Laboratory data on admission showed a white blood cell count of 1,580/microliter, a hemoglobin level of 9.1 g/dl, and a platelet count of 113 x 10(3)/microliter. A small percentage of abnormal mononuclear cells were present in the peripheral blood. A bone marrow biopsy specimen demonstrated myelofibrosis and diffuse infiltration of abnormal monoculear cells with a mature B cell phenotype. A bone marrow aspirate showed 29% abnormal mononuclear cells, which had an indented or folded nucleus and reticular nuclear chromatin. Moderate to strong tartarate-resistant acid phosphatase activity was detected in these cells. Although the cytoplasmic projections were poorly preserved in specimens stained with May-Giemsa, fresh preparations showed numerous slender cytoplasmic projections by phase-contrast microscopy. The hairy cells had the phenotype CD5-, CD10-, CD11c+, CD19+, CD20+, CD25+, lambda. The patient was diagnosed as having European-American-type hairy cell leukemia (HCL) without splenomegaly, which is quite rare in Japan. The value of phase-contrast microscopy for recognition of the hairy cells was emphasized. The patient was treated successfully with deoxycoformycin (DCF).

Antibiotics, Antineoplastic↗
PubMed2004

[Value of urinary sediment cytology in evaluation of acute tubular necrosis after kidney transplantation].

AIM: The aim of the study was to assess the clinical value of urinary sediment cytology (USC) by use of phase-contrast microscopy in the evaluation of acute tubular necrosis (ATN) during the early period after kidney transplantation. The study was performed at the Cytology Laboratory, Department of Nephrology and Dialysis, Clinical Hospital Center Rijeka, Croatia. PATIENTS AND METHODS: Patients included 141 kidney recipients, 99 male and 42 female, mean age 40 +/- 13 (range 8-72) years, who had received kidney allograft during the period of ten years, and who were treated at the University Department of Internal Medicine, Rijeka Clinical Hospital Center. The majority of patients (76%) had received cadaveric kidneys. Urinary sediment was analyzed for the presence of renal tubular cells, isomorphic erythrocytes, lymphocytes, casts and debris. Renal tubular cells on USC were recognized as the most constant sign of ATN. The presence of lymphocytes should arise suspicion of rejection. MAIN OUTCOME MEASURES: A typical cytologic profile of acute tubular lesion consists of tubular cells, isomorphic erythrocytes, casts, cellular and/or amorphic debris. RESULTS: USC by use of phase contrast microscopy is confirmed as a method of a very high sensitivity (82%) and specificity (93%) in the evaluation of ATN in transplanted kidney patients during early post-transplantation period. In situations of coexistence of several causes of allograft dysfunction, "mixed" cytologic pictures were frequently created, from which it is difficult or almost impossible to identify the actual cause of kidney dysfunction. In these cases, the final judgment should be made solely by histologic evaluation, which still represents the gold standard in the evaluation of kidney allograft dysfunction. CONCLUSION: Serial USC, when thoroughly examined using phase-contrast microscopy, is a simple, noninvasive, fast, easily repeatable and inexpensive diagnostic method of high sensitivity and specificity in the evaluation of ATN during the early phase after kidney transplantation.

Adolescent↗