Optical tracking and detection of immunomagnetically selected and aligned cells.

We have developed a platform for cell analysis based on immunomagnetic selection and magnetic alignment of cells in combination with an epi-illumination tracking and detection system. Whole blood was labeled with ferromagnetic nanoparticles and fluorescent probes, and placed in a magnetic field in a chamber. Cells labeled with ferromagnetic nanoparticles moved upward and aligned along ferromagnetic lines deposited by lithographic techniques on an optically transparent surface of the chamber. An epi-illumination system using a 635 nm laser diode as a light source scanned the lines and measured signals obtained from the aligned cells. The cell counts per unit of blood volume obtained with the system correlated well with those obtained from the counts from a standard hematology analyzer and flow cytometer. The cell analysis platform is significantly less complex and more sensitive than current cell analysis equipment and provides additional functionality through its ability to subject the cells to repeated and varied analyses while they remain in a natural environment (i.e., whole blood).

On the mechanism of Clq binding to antibody-I. aggregation and/or distortion of IgG vs combining site-transmitted effects.

It was shown previously that in sheep calcium-dependent anti-GAT there is a subpopulation which reacts with and can be precipitated with poly G [Liberti (1975) Immunochemistry 12, 303-310]. This entire subpopulation was also found to react with the cross-reacting polypeptides GA and GT. Furthermore, from hydrogen exchange experiments, it was found that only the immunizing antigen GAT completely filled the combining sites of these antibodies whereas poly G was shown to occupy an average of 47% of all sites, and GA and GT, 75 and 85% respectively. Since precipitins formed with this subpopulation and each of these antigens should have reasonably similar densities and orientations of 'aggregated' IgG but differing combining sites occupancies, we have used this system to explore the relative role of Fc aggregation and/or IgG distortion vs combining site-transmitted effects on the binding of Clq to antibody. For two preparations of this subpopulation (one of high avidity, the other obtained via poly G-Sepharose and of lower avidity) there is only a 5% difference in the delta G (10.2-10.8 kcal/mole) of Clq-IgG interaction for a change in combining site occupancy of 47-100%. For the high-avidity preparation there is a correlation between delta G and degree of ligand occupancy of combining site. This could reflect combining site-transmitted effects or may be related to small differences in the molecular architecture of these precipitins. Clq saturation curves support the latter notion. In view of the very moderate effect of combining site filling (from 47 to 100%) on Clq-IgG interaction for the high-avidity preparation and the absence of any correlation for the lower-avidity preparation. It appears that an allosteric model for antibody initiation of complement is untenable. Unless combining site-originating contributions are completed when less than 50% of an antibody-binding site is occupied by ligand, it would seem that Clq binding to immune complexes must be governed either by enhanced interactions resulting from Fc clustering which occurs via antibody interactions with antigen or by distortion of the antibody molecular upon ligand binding or some combination thereof.

Isolation and characterization of highly stable rabbit C1q.

Rabbit C1q has been isolated by techniques which retain elements of previous isolation methods along with novel procedures incorporated to yield stable, non-aggregating, highly purified and biologically active C1q. The procedure involves euglobulin preparation by dialysis of serum and two column chromatographies, DEAE-Sephadex and Ultragel ACA 34. The method results in yields of C1q from rabbit sera of 20-30%. Ouchterlony analysis, sedimentation velocity experiments, equilibrium ultracentrifuge molecular weight analysis, SDS, polyacrylamide gel electrophoresis and isoelectric focusing experiments indicate the presence of only a single component, which has all the physical characteristics previously reported for C1q. The preparation has all the biological characteristics of C1q such as the ability to agglutinate IgG-coated latex particles and to restore hemolytic activity to RC1q serum. Preparations obtained by this procedure are stable to freezing (-70 degrees C) for extended periods of time and show no evidence of aggregation upon thawing following periods of storage of up to 6 months. Mild acetylation and handling of preparations for extended periods at 37 degrees C and 45 degrees C indicate no evidence of aggregation or loss of agglutinating and hemolytic activities. Preparations so isolated meet all the criteria required for critical physical-chemical studies of this protein.

A surface-labeled 18 kilodalton antigen in Schistosoma mansoni.

A mild surface-labeling procedure was applied to various developmental stages of Schistosoma mansoni. An 18 kDa protein was preferentially labeled in freshly transformed schistosomula. The labeled protein was equally present on skin-penetrated and mechanically prepared schistosomula and it disappeared upon digestion of intact parasites with proteolytic enzymes. The 18 kDa protein could be specifically precipitated with an antiserum raised against 3-h schistosomula. Six-day lung forms also presented a single major labeled protein component, but the apparent molecular weight of this protein in acrylamide gels was higher than 18 000. Fourteen-day-old and adult schistosomes showed only weak labeling distributed in several bands. The radioactivity pattern of adult worms (but not of schistosomula) could also be obtained by incubating fresh parasites in a medium which had previously been used to label schistosomes and to which a 100 000-fold excess of 127I over 125I had been added. Post-labeling incubation of parasites was found to be essential for the detection of stable surface proteins.

Magnetic resonance imaging of abscesses using lipid-coated iron oxide particles.

RATIONALE AND OBJECTIVES: The authors investigated whether iron oxide particles can be used as a magnetic resonance imaging (MRI) contrast agent to image abscesses in a two-stage experimental design. METHODS: Human buffy coat was incubated with iron oxide particles of different sizes and coatings. Smears of the incubation mixture were made on a glass slide and stained for iron. The percentage of iron oxide uptake was determined by counting 100 neutrophils and monocytes and scoring the number of cells that contain iron. Subcutaneous abscesses were created in the flanks of 18 Sprague-Dawley rats by injecting them with 0.1 mL of turpentine. Iron oxide was given intravenously, and the animals were imaged by MRI (1.5 T) 12 to 24 hours later. Different iron oxide coatings and doses were compared. RESULTS: The four different types of coating (constant fragment [Fc] of IgG, bovine serum albumin [BSA], lipid [Ferrosome], and dextran) had an uptake of 72% +/- 5.3%, 61% +/- 6.2%, 30.5% +/- 6.8%, and 5% +/- 2.5%, respectively. Comparison of two particle sizes (mean, 90 versus 35 nm) showed the large particles to have higher uptake (61% +/- 6.2%) compared with the small particles (6% +/- 1.8%) (P less than .001). Post-contrast imaging of the rats showed a hypointense ring around the abscess only in the animals injected with the lipid-coated agent. The effect was discernible within 12 hours after contrast injection and at a dose of 25 mumols iron/kg. Histologic sections showed phagocytic cells with iron granules in the periphery of the abscess. No hypointense ring on MRI or iron granules on histologic sections was seen around the abscess of the control animals or those injected with BSA-iron oxide or Fc-iron oxide. CONCLUSIONS: Lipid-coated iron oxide particles can be used to image abscesses by virtue of their phagocytosis into surrounding inflammatory cells. Positive uptake of these particles by human phagocytes in vitro suggests that similar results may be applicable in humans.

A model system illustrating the isolation and enrichment of a rare population of tumour cells in bone marrow.

A model system employing a modified nylon matrix is described for the separation of rare cells titrated into either a leukaemic cell line or normal bone marrow. A 75- to 125-fold enrichment and recovery of the rare cell population was achieved, starting from an initial level of 0.014 to 0.2% of the total population. The rare cell population was identified by pre-labelling with Hoechst 33342, which intercalates into the DNA, and renders cells highly fluorescent. Separation and recovery of cells was totally dependent on the use of a panel of monoclonal antibodies binding to the labelled population. The nylon matrix, precoated with an anti-mouse immunoglobulin, traps the cells coated with monoclonal antibodies, and these can be released simply by gentle manipulation of the matrix. The matrix employed has been shown to not specifically trap committed bone marrow progenitors as determined by CFU-GM, BFU-E and CFU-GEMM assays. The use of this technique should simplify the isolation of rare tumour cells metastasizing to bone marrow.

Quantitative immunochromatographic analysis: theory and application to theophylline immunoassay.

The development of an immunochromatographic technique suitable for rapid analysis of biological fluids is described. Quasi-one-dimensional antibody lattices specific for theophylline were constructed by packing Sepharose beads conjugated with specific antibody into specially designed narrow capillary tubes. The design of these capillary columns was such that they would subtract a preset threshold quantity of antigen (label and analyte) from the total amount presented. Labeled antigen, which appeared in the flowthrough, could then be used to precisely quantitate the analyte present. The ideal format would permit very precise subtraction of 100% of the available antigen up to the threshold amount and none of the remainder. The microcolumn described here comes close to this ideal behavior through the attainment of very high ratios of bound/free antigen. The elevated bound/free ratio could be explained by theoretical analysis of the effect on equilibria of the high antibody concentration in this quasi-one-dimensional system. Lattices containing anti-theophylline antibodies were used to develop a competitive enzyme immunoassay for theophylline which demonstrated a dose-response that was closely similar to that predicted by theoretical treatment. The entire assay procedure was performed in less than 30 min and demonstrated a sensitivity limit of approximately 20 ng/ml. Preliminary studies on clinical serum samples suggest that this assay has potential for the routine analysis of biological fluids.

Minimum estimate of the lifetime of histone messenger RNA of HeLa cells.

The lifetime of histone mRNA of HeLa cells has been studied by its kinetic of approach to steady-state labeling. Cells preincubated with low concentrations of actinomycin D to inhibit rRNA synthesis, were incubated with ((3)H)uridine. Linear incorporation of uridine was observed for only two hours under the conditions chosen. Polyribosomes were isolated from cells incubated overnight with trace amounts of ((14)C)uridine and for 30 to 150 min with ((3)H)uridine. RNA was extracted from polyribosomes and fractionated by polyacrylamide gel electrophoresis. Histone mRNA was identified as a peak migrating in a characteristic position, which was absent in gels of RNA obtained from cells treated with the inhibitor of DNA synthesis cytosine arabinoside. The kinetic of labeling of histone mRNA was linear up to 150 min, which represents a minimum estimate of the lifetime of this mRNA.

Immunomagnetic colloids for the enrichment of tumor cells from peripheral blood and bone marrow: a model system.

The characterization of tumor cells in bone marrow harvested for autologous bone marrow rescue remains a problem due to the limited sensitivities of the techniques available for their analysis. This complicates the use of purging techniques as their usefulness is debated, specifically because it is not known if they can remove all residual tumor cells from either peripheral blood stem cell harvests or bone marrow. Apart from developing more sensitive techniques for tumor detection, one way of increasing our efficiency at identifying rare malignant cells in normal hematopoietic cells is to develop approaches to enrich the population of interest prior to analysis. Here, we describe a laboratory-based system for tumour enrichment employing panels of monoclonal antibodies (MAbs) and an immunomagnetic colloid. In model systems, tumor cells could be enriched approximately 100-fold with high yield and purity. The adaptations of this technology to permit further cell enrichment and the choice of either an immunological technique or a molecular approach to tumor identification are discussed in detail.

Solvent effects on the structure of rabbit Clq, a subcomponent of the first component of complement.

The effect of solvent conditions on the conformation of rabbit Clq was studied by both spectroscopic and nonspectroscopic methods. The conformation of Clq in buffered saline solutions at pH 7.4 or 6.0 did not differ significantly from Clq at twice the saline concentration as determined with circular dichroism, difference spectroscopy, and tritium-hydrogen exchange techniques. Addition of calcium to the buffers had no structural effects in any of the conditions examined. Hydrogen exchange experiments performed at pH 7.4 were also unaffected by magnesium, manganese, or ethylenediaminetetraacetic acid. With all the methods used a pH effect was observable between 5.1 and 8.3. From solvent perturbation difference spectroscopy results it was calculated that the equivalent of 10 +/- 2 and 6 +/- 1 mol of tyrosine and tryptophan/mol of Clq, respectively, became exposed at the lower pH. A small positive CD band in the 231 to 235 nm region decreased in wavelength and increased in magnitude as a function of decreasing pH, indicating tyrosine exposure at the lower pH and possibly changes in the collagen-like structure of Clq. Hydrogen exchange experiments indicate a small, but significant, conformation transition occurring in the pH 5 region and a stabilization of conformation between pH 6 to 8. From these results the conformational pH dependence was interpreted as an acid expansion of Clq with a minor conformational transition occurring between pH 5 AND 6. These effects may in part be associated with decreased Clq-Ig interactions which have been observed at the lower pH.

Stage-specific expression of a Schistosoma mansoni polypeptide similar to the vertebrate regulatory protein stathmin.

The ubiquitous vertebrate protein stathmin is expressed and phosphorylated in response to a variety of external and internal signals. Stathmin, in turn, controls cell growth and differentiation through its capacity to regulate microtubule assembly dynamics. This is the first report on the molecular cloning and characterization of a stathmin-like protein (SmSLP) in an invertebrate, the human blood fluke Schistosoma mansoni. SmSLP is first synthesized at high levels in the intermediate molluscan host and completely disappears 48 h after penetration into the mammalian host. The protein is preferentially iodinated in intact immature parasites using the Bolton-Hunter reagent, can be quantitatively extracted in high salt buffers, and remains soluble after boiling. Native SmSLP was partially sequenced, and its complete structure was derived from the cloning and sequencing of its cDNA. The sequence is up to 26% identical to vertebrate stathmin sequences and contains two potential phosphorylation sites. Native SmSLP is indeed phosphorylated because phosphatase digestion shifts its mobility in electrofocusing gels. SmSLP associates with tubulin, as suggested by immune co-precipitation results. In vitro experiments demonstrated that SmSLP inhibits tubulin assembly and causes the depolymerization of preassembled microtubules, thus probably fulfilling regulatory roles in critical steps of schistosome development.

Cell analysis system based on immunomagnetic cell selection and alignment followed by immunofluorescent analysis using compact disk technologies.

BACKGROUND: Although the flow cytometer has become the standard in cell analysis, it has limitations. Recently, we introduced a new cell analysis method based on immunomagnetic selection and aligning of cells. No flow system is needed and cell analysis can be performed in whole blood. METHODS: Whole blood is incubated with fluorescent labels and immunomagnetic nanoparticles. The blood is injected into a capillary that is in a strong magnetic field. The immunomagnetic-labeled cells move upward and align themselves along ferromagnetic lines present on the upper surface of the capillary. An optical focus and tracking system analogous to that used in a conventional compact disk player focuses a 635-nm laser-diode on the magnetically aligned cells. The emitted fluorescence signals are projected on two photomultipliers. Allophycocyanin (APC)-labeled CD4 (CD4-APC) and Cyanin5.5 (Cy5.5)-labeled CD8 (CD8-Cy5.5) antibodies and Oxazine750, all red excited, are used as fluorescent labels. RESULTS: A differential white blood cell count performed in whole blood is obtained using the CD4-APC in combination with Oxazine750. The results are compared with the Technicon-H1 hematology analyzer. Correlation coefficients of 0.91 for neutrophilic granulocytes, 0.93 for lymphocytes, 0.93 for monocytes, and 0.96 for eosinophilic granulocytes were obtained. Immunofluorescence is demonstrated using CD4-APC and CD8-Cy5.5. The absolute counts obtained for CD4+ and CD8+ are compared with the Coulter Epics XL flow cytometer. Correlation coefficients of, respectively, 0.91 and 0.94 were obtained. CONCLUSION: We conclude that our system is as capable as a standard flow cytometer or hematology analyzer for a reliable routine white blood cell analysis, including immunophenotyping, and can be used as an easy-to-handle disposable white blood cell test.

Correlation between chronic prostatitis syndrome and pelvic venous disease: a survey of 2,554 urologic outpatients.

OBJECTIVES: In this study we evaluated the association between chronic prostatitis syndrome (CPS), varicocele and hemorrhoids as manifestations of a pelvic venous disease. METHODS: Our retrospective study was based upon 2,554 patients treated in two general urology clinics over the past 10 years. We have assessed the incidence of CPS among urological patients. RESULTS: We found 483 patients with CPS, representing 18.9% of the total number of visits at the outpatient clinic. In this group the percentage of varicocele and hemorrhoids was 14.69 and 8.48%, whereas in a control group these figures were 5.02 and 5.84%, respectively (p<0.001 and 0.1054). Such a difference is statistically significant and suggests a higher prevalence of varicocele in the CPS group, but this may be due to a methodological error of the retrospective study. CONCLUSION: Only a prospective study, which is of importance due to the frequency of the disease, can give a precise answer to this question.