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Current analytical methodology for iodopropynyl butylcarbamate (IPBC) analysis focuses on the use of liquid chromatography and mass spectrometer (LC-MS), but the high instrumentation and operator investment required has resulted in the need for a cost effective alternative methodology. Past publications investigating gas chromatography with electron capture detector (GC-ECD) for IPBC quantitation proved largely unsuccessful, likely due to the preservatives limited thermal stability. The use of pulsed injection techniques commonly used for trace analysis of thermally labile pharmaceutical compounds was successfully adapted for IPBC analysis and utilizes the selectivity of GC-ECD analysis. System optimization and sample preparation improvements resulted in substantial performance and reproducibility gains. Cosmetic formulations preserved with IPBC (50-100ppm) were solvated in toluene/isopropyl alcohol and quantified over the 0.3-1.3μg/ml calibration range. The methodology was robust (relative standard deviation 4%), accurate (98% recovery), and sensitive (limit of detection 0.25ng/ml) for use in routine testing of cosmetic formulation preservation.

In the past, ablation of cancer cells using radiofrequency heating techniques has been demonstrated, but the current methodology has many flaws, including inconsistent tumor ablation and significant ablation of normal cells. Other researchers have begun to develop a treatment that is more selective for cancer cells using metallic nanoparticles and constant electric field exposure. In these studies, cell necrosis is induced by heating antibody functionalized metallic nanoparticles attached to cancer cells. Our approach to studying this phenomenon is to use similarly functionalized metallic nanoparticles that are specific for the T47D breast cancer cell line, exposing these nanoparticle cell conjugates to a nanosecond pulsed electric field. Using fluorescent, polystyrene-coated, iron-oxide nanoparticles, the results of our pilot study indicated that we were able to ablate up to approximately 80% of the cells using 60 ns pulses in increasing numbers of pulses and up to approximately 90% of the cells using 300 ns pulses in increasing numbers of pulses. These quantities of ablated cells were achieved using a cumulative exposure time 6 orders of magnitude less than most in vitro constant electric field studies.

An analytical method has been developed for the determination of alcohol ethoxylate (AE) components in sewage sludge. The method has been extensively ring tested in several industrial laboratories and concentrations in sludge samples from a number of EU countries have been obtained. The method is based on a methanol soxhlet extraction of centrifuged sludge, which is then cleaned up using an alumina column, followed by derivatisation with naphthoyl chloride and a further alumina column clean-up. The extract is analysed using high performance liquid chromatography (HPLC) with fluorescence detection. The method is capable of determining alcohol ethoxylate components in the range of C12–C18 alkyl chain lengths with an ethoxylate chain of EO4 up to approximately EO20 in sludge samples. The detection limit is approximately 20–30 mg kg−1 of total AEs in dry weight of sludge. Using the method, sludges from several European Sewage Treatment Plants (STP) were analysed. The concentration of the AEs, which are primarily linear, in digestor inlet averaged 1164 mg kg−1 (550–2947 mg kg−1) and in outlet sludges the mean value obtained was 167 mg kg−1 (−1). At those plants in which concentrations were monitored in both inlet and outlet sludges, removal of the AEs by anaerobic digestion at the STP averaged 82% (range 61–93%). The interlaboratory relative standard deviation of the procedure was around 40% for the digester sludges analysed. The method developed provides a more accurate estimate of the environmental level of AE components compared to existing colorimetric approaches, but the method will over-estimate the concentration of alcohol ethoxylates in sludges due to the non-specific nature of the detection. However, it is sufficiently robust and accurate to estimate alcohol ethoxylates in sludges and hence concentrations that could be applied to soil.

Using phenanthrene as a model analyte, we show that extraction from aqueous solution is possible via adsorption onto the surface of a poly(tetrafluoroethylene)-coated magnetic stir bar. Desorption from the stir-bar via ultrasonic agitation in acetonitrile solution allows quantitation of the analyte in solution. The technique is presented as a simple strategy, using commonly available laboratory apparatus, for the extraction of analytes from solution based on a surface adsorption processes: stir-bar adsorptive extraction (SBAE).

This paper describes the clinical orthodontic treatment of two cases that were awarded the British Orthodontic Society Membership in Orthodontics Prize.

The supercritical fluid extraction (SFE) of metal ions was assessed in terms of the ability of the process to extract, solvate, transport and collect metal species. The extraction efficiency (per cent removed from the sample matrix), collection efficiency (per cent recovered in the collection solvent) and SFE residue (unextracted metal complex or metal ion recovered from the apparatus by flushing with an organic or acidic solvent, respectively) were determined to ascertain whether a mass balance for the metal could be achieved. Using this approach, it was demonstrated that the amount of metal extracted from the sample matrix did not necessarily correspond to the concentration of metal recovered in the collection solvent, as the chelated metal complex could partially degrade and/or dissociate in the supercritical fluid prior to collection. Typically, a discrepancy of 1 to 15% could be obtained. The non-fluorinated metal β-diketones and dithiocarbamates had a greater tendency to dissociate and/or degrade in a supercritical fluid (i.e., ca. 14 and 5%, respectively) than the corresponding fluorinated metal complex (i.e., ca. 6 and 1%, respectively). As for the collection solvents, a range of suitable fluids were found to be capable of obtaining quantitative metal complex recoveries, including methanol, chloroform, isobutyl methyl ketone or 10% nitric acid. A radioactive isotope, 177Lu, was used to trace the location of the metal residue in the SFE apparatus.

The retention behavior in capillary supercritical fluid chromatography (SFC) of a range of ferrocene derivatives is demonstrated. Prediction of retention times and hence elution order of the ferrocene derivatives based on a knowledge of solute molar volume, solute solubility parameter and the physical parameters of the chromatography column system is shown to be feasible using this procedure.

Supercritical fluid extraction of cobalt ions was evaluated in terms of the ability of the process to extract, solvate, transport and to collect the metal species. The aim of the study was to ensure that a mass balance could be obtained for the metal before and after extraction with supercritical carbon dioxide (CO2). The stability of the ligand and resulting metal complex were assessed as was the synergistic effect of the presence of water in the extraction. Two widely used β-diketones, namely, acetylacetone (AcAc) and hexafluoroacetylacetone (HFAcAc) were chosen to extract spiked cobalt from the surface of stainless steel and >90% of the metal was extracted and collected using the HFAcAc ligand with supercritical CO2.

The solubility of 49 metal-containing compounds and 15 “free” ligands in supercritical fluid carbon dioxide is reviewed. Solubilities were found to range over eight orders of magnitude, with the highest value of solubility being 56 g l−1. Metals complexed with fluorine substituted ligands were found to be the most soluble, and metals complexed with phenyl-substituted ligands the least soluble. A general trend for increasing solubility with increasing oxidation state is observed and this is correlated with the increased number of coordinating ligands protecting the metal center from interaction with the supercritical fluid and also due to the increased number of solvation interactions with the increased number of ligand groups.

The deacidification of high-acidity oils from Black cumin seeds (Nigella sativa) was investigated with supercritical carbon dioxide at two temperatures (40 and 60°C), pressures (15 and 20 MPa) and polarities (pure CO2 and CO2/10% MeOH). For pure CO2 at a relatively low pressure (15 MPa) and relatively high temperature (60°C), the deacidification of a highacidity (37.7 wt% free fatty acid) oil to a low-acidity (7.8 wt% free fatty acid) oil was achieved. The free fatty acids were quantitatively (90 wt%) extracted from the oil and left the majority (77 wt%) of the valuable neutral oils in the seed to be recovered at a later stage by using a higher extraction pressure. By reducing the extraction temperature to 40°C, increasing the extraction pressure to 20 MPa, or increasing the polarity of the supercritical fluid via the addition of a methanol modifier, the selectivity of the extraction was significantly reduced; the amount of neutral oil that co-extracted with the free fatty acids was increased from 23 to 94 wt%.

Real world samples which contain high concentrations of water and/or extractable material frequently cause intermittent or irreversible plugging of the flow control device during off-line supercritical fluid extraction (SFE). A supercritical fluid (SF) flow control/collection device has been developed which can simultaneously maintain the extraction flow-rate of the SF (±0.1 ml/min) and quantitatively (>90%) collect analytes as volatile as n-octane directly into an organic solvent. With this device, the extract is partially depressurized through a heated capillary restrictor and into a pressurized collection solvent, so that both temperature and pressure are used to maintain the solubility of the extract in the SF. The pressurized mixture is finally depressurized to atmospheric conditions using a backpressure regulator, so the extract can be recovered in the collection vial. Depending on the sample matrix, a restrictor heater temperature of 200 °C and a backpressure regulator with a heated (>5 wt% water in sample) or unheated (

Supercritical fluid extraction of metals in the presence of complexing agents for environmental monitoring, cleanup, and metals processing is now being extensively researched, and there is a need for solubility data of metal complexes. In this paper, the method used is the combination of a study of chromatographic retention over a wide range of pressures at 40, 50, 60, and 70 `C, followed by selected direct solubility measurements, to provide a comprehensive set of solubility results obtained with minimum effort. The method relies on the assumption that an inverse relationship exists between chromatographic retention and solubility in the mobile phase. This assumption is checked in this study. Results are presented for the solubility of ferrocene in supercritical carbon dioxide and a nickel complex, (5,7,12,14-tetramethyl-2,3:9,10-dibenzo[b,i][1,4,8,11]tetraazacyclotetradecine)nickel(II), in supercritical carbon dioxide modified with 10 vol % methanol.

Summary form only given. Invasive ablation of cancer cells using radiofrequency-heating techniques has been demonstrated clinically, but the current methodology still has many flaws, including inconsistent tumor ablation, and significant healthy cell death1. Research has been done to develop a method that is both non-invasive and more selective for the cancer cells using metallic nanoparticles and constant electric field exposure2,3. The mechanism by which cell necrosis is achieved in these studies is the heating of functionalized metallic nanoparticles, which are attached to the cancer cells via antibody conjugation. Our approach to studying this phenomenon is to use similarly functionalized metallic nanoparticles that are specific for the T47D breast cancer cell line, but expose these nanoparticle-cell conjugates to a nanosecond pulsed electric field. The percentage of cells killed from hyperthermia can then be assessed using hemacytometer cell staining and counting techniques.

A coupled supercritical fluid extraction-gas chromatography (SFE-GC) method has been developed for the quantitative extraction and analysis of gasoline and diesel range organics from real world environmental samples. Petroleum-contaminated samples containing gasoline- to diesel- and motor oil-range hydrocarbons (total hydrocarbon content typically ranging from 2 to 26 mgg) could be quantitatively extracted by a 15-min SFE-GC extraction using 400 atm (1 atm = 101 325 Pa), 60°C CO2. The SFE-GC hydrocarbon recoveries from real-world samples were comparable to those obtained by sonicating the samples in methylene chloride for 14 h, except for the gasoline recovery which was higher by SFE-GC analysis due to the more efficient collection of the more volatile analytes. Reproducibilities for replicate SFE-GC extractions and analyses were typically

Of 21 potential drying agents investigated, five (anhydrous and monohydrated magnesium sulfate, molecular sieves 3A and 5A and Hydromatrix) were able successfully to prevent restrictor plugging by water during off-line supercritical fluid extraction (e.g., 400 atm CO2 at 60°C) by retaining the majority of the water (but generally not the analytes of interest) in the extraction cell. Increasing the extraction temperature (e.g., to 150°C) or adding a polar modifier [10% (v/v) methanol] to the CO2 extraction fluid greatly reduced the amount of water the drying agents retained. However, when 10% (v/v) toluene was used for the extraction, the drying agents were able to retain the majority of the water (ca. 80% w/w). Polar and non-polar pollutants were quantitatively extracted from the wet drying agents (i.e., water present), but nearly all of the drying agents selectively retained at least one of the polar analytes if used dry (i.e., no water present), thus demonstrating the need for a spike recovery study to determine the potential for analyte loss. The successful drying agents eliminated restrictor plugging when used with moderately wet [ca. 20% (w/w) water at a 1:1 reagent-to-sample ratio] and very wet [ca. 90% (w/w) water at 4:1 reagent-to-sample ratio] samples without the need to heat the restrictor or the collection solvent.

Fused-silica restrictors used for off-line supercritical fluid extraction (SFE) frequently break when extractions are performed with polar supercritica

The development of quantitative supercritical fluid extraction (SFE) methods for the recovery of organic pollutants from environmental samples requires three steps: quantitative partitioning of the analytes from the sample into the extraction fluid, quantitative removal from the extraction vessel, and quantitative collection of the extracted analytes. While spike recovery studies are an excellent method to develop the final two steps, they are often not valid for determining extraction efficiencies from complex real-world samples such as soils and sediments, exchaust particulates, and sludges. SFE conditions that yield quantitative recoveries of spiked analytes may recover < 10% of the same analytes from real-world samples, because spiked pollutants are not exposed to the same active sites as the native pollutants. Because of the heterogeneous nature of environmental samples, the partitioning step may be controlled by analyte solubility in the extraction fluid, kinetic limitations, and/or the ability of the extraction fluid to interrupt matrix-analyte interactions. While the interactions that control SFE rates from heterogenous environmental samples are not well understood, a generalized scheme for developing quantitative SFE methods is proposed based on interactive considerations of the collection efficiencies after SFE, fluid flow parameters in the extraction cell, analyte solubility, extraction kinetics, and analyte-matrix-extraction fluid interactions. The proposed development scheme includes increasing SFE extraction rates by the use of more polar fluids than CO2 such as CHClF2, the addition of organic modifiers to CO 2, and the use of high temperature extractions with pure CO2. Validation of quantitative extractions based on multiple extraction methods (SFE followed by liquid solvent extractions) is also described.

The relative extraction rates of native polycyclic aromatic hydrocarbons (PAHs) ranging from naphthalene (M = 128) to benzo[b]fluoranthene (M = 252) and those of spiked deuterated PAHs (d-PAHs) from heterogeneous environmental samples including petroleum waste sludge, urban air particulate matter (SRM 1649), and railroad bed soil were compared using sequential extractions with pure supercritical CO[sub 2] or modified (10% v/v methanol) supercritical CO[sub 2] and using sonication with methylene chloride. Regardless of the spiking method (injection of the spike or suspension of the sample in the spiking solution) or aging time (up to 14 h), the extraction rates of most of the spiked d-PAHs were substantially (up to 10-fold) higher than those of the same native PAHs. Differences in extraction rates of the spiked and native PAHs were most dramatic for the lower molecular weight PAHs, indicating that relatively volatile species such as naphthalene must be tightly bound in order to remain associated with a real-world sample. In most cases, 30-min extractions with pure CO[sub 2] quantitatively recovered (>90%) the spiked deuterated-PAHs, but only extracted ca.25-80% of the native PAHs. Similar differences were observed using conventional methylene chloride sonication, demonstrating that spike recovery studies are not valid for developing quantitativemore » extraction methods for heterogeneous environmental samples. 21 refs., 8 figs., 4 tabs.« less

Smith, R.M. and Burford, M.D., 1993. Comparison of flavanoids in feverfew varieties and related species by principal components analysis. Chemometrics and Intelligent Laboratory Systems, 18: 285–291. The fingerprint patterns of the high-performance liquid chromatographic separations of the flavanoids from 106 feverfew [Tanacetum parthenium (L.) Schultz Bip.], tansy (Tanacetum vulgare L.) and German chamomile (Matricaria chamomilla L.) plants and four commercial feverfew products were correlated by principal components analysis. It was possible to distinguish the three medicinal herb species by using the first, second and third principal components and to demonstrate that the commercial products contained feverfew.

The High Operating Temperature Auger suppressed infrared detector concept is being pursued using the high density vertically integrated photodiode (HDVIP ® ) architecture and an n + -p device structure. Dark current densities as low as 2.5 mA/cm 2 normalized to a 5 µm cutoff at 250K have been demonstrated on these diodes. These dark currents imply minority carrier lifetimes in excess of 300µsec. 1/f noise in these devices arises from the tunneling of charge into the passivation interface, giving rise to a modulation in the surface positive charge and hence to the width of the depletion region in the p-side of the device and a modulation in the total dark current. The measured 1/f noise is in agreement with the predictions of this model, with very low noise being observed when the lifetimes are high. Keywords: Mercury cadmium telluride, infrared detector, focal plane array, high operating temperature, HOT 1. INTRODUCTION 1.1 Motivation In the past, most photon detecting infrared Focal Plane Arrays (FPAs) have operated at a temperature of 77K or below. There is a considerable system advantage if the operating temperature of the FPA can be increased, namely that the resulting Dewar and Cooler package into which the FPA is integrated will be considerably lighter, smaller, and thus cheaper. In addition the power required to cool the FPA can be decrease by a factor of ~5-10, as illustrated in Figure 1. Implementation of this technology enables new system concepts and functionality.

Supercritical carbon dioxide was used to extract the sesquiterpene lactone parthenolide, which is reported to be the active component of the medicinal herb feverfew ( Tanacetum parthenium ), from dried plant material for gas-liquid chromatographic analysis. The extracts also contained significant amounts of camphor and chrysanthenol acetate. The pressure and densities required for quantitative extraction and the methods of sample collection were studied. The addition of methanol or acetonitrile as modifiers in the carbon dioxide gave higher yields of parthenolide bu the extractions were less selective and considerable amounts of co-extractives were obtained. By employing a trapping column made of cellulose or silica the purity of the extracts could be improved and a practical method for the rapid isolation of parthenolide is described.

Real-world environmental samples which contain high concentrations of water and/or extractable matrix components frequently cause intermittent or irreversible plugging of capillary flow restrictors during off-line supercritical fluid extraction. Heating the entire restrictor at 50°C produces a constant extraction flow-rate (± 0.03 ml/min) for such samples, but poor collection efficiencies (30–65% recovery for polycyclic aromatic hydrocarbons) were obtained because the supercritical fluid could not be depressurized directly into the collection solvent. While the collection efficiency was improved (80–90%) by nebulizing an organic solvent with the restrictor effluent, a simpler method was to heat all but the last 3 cm of the restrictor and to depressurize the extract directly in the collection solvent. Depending on the sample matrix, restrictor heater temperatures ranging from 50 to 200°C were required to avoid restrictor plugging. With proper heating, constant extraction flow-rates (± 0.07 ml/min) and high collection efficiencies (90–100%) for polycyclic aromatic hydrocarbons and n -alkanes as volatile as n -octane were achieved.

Supercritical fluid extraction (SFE) recoveries for native pollutants including PCBs from a standard reference material sediment (SRM 1939), PAHs from a petroleum waste sludge, and PAHs from railroad bed soil were compared using supercritical CHCIF 2 (Freon-22), N 2 O, and CO 2 . While SFE with pure CO 2 yielded the lowest recoveries for each sample, CHCIF 2 consistently yielded the highest extraction efficiencies, most likely because of its high dipole moment

α-Cellulose has been used as a model plant matrix to investigate the conditions required to optimize the supercritical fluid extraction of typical plant constituents, limonene, caryophyllene, carvone, eugenol and santonin, using carbon dioxide as the extraction medium. The conditions required for the successful recovery of the analytes were monitored by gas-liquid chromatography. Timed recovery studies enabled differences in the rates of extraction to be determined to ensure that sufficiently long extraction runs were used. Subcritical and supercritical extractions over the ranges −10 to 80°C and 50–250 bar were examined and 250 bar and 40°C were chosen as the optimum conditions. The effects of the addition of modifiers to the supercritical fluid were also examined. The work also demonstrated that increased selectivity for polar analytes such as lactones could be obtained by trapping the extract on a silica column coupled with selective elution.

Supercritical fluid extraction (SFE) collection efficiences of 66 compounds with a wide range of volatility and polarity were examined. Good collection efficiencies required efficient partitioning of the analyte into the collection solvent after depressurization, and factors including collection solvent polarity and temperature were found to be more important than collection solvent volume and height. Heating the collection solvent with a heat gun to avoid plugging of the outlet restrictor resulted in 20−50% losses of the more volatile analytes, while > 90% trapping of all test analytes could be attained by controlling the solvent temperature at 5°C. Extraction cell geometry (“long, narrow” versus “short, broad” vessel) at constant internal volume and the orientation of the extraction cell were found to have negligible effects on the extraction rates of polycyclic aromatic hydrocarbons (PAHs) from railroad bed soil and flavor and fragrance compounds from lemon peels. The supercritical fluid flow-rate also had little effect on the extraction rate of native PAHs provided that it was sufficient to sweep the cell dead volume every ca. 3 min.

Conventional supercritical fluid extraction (SFE) system are constructed using stainless steel due to its relative inertness, ready availability, tensile strength and low cost. Recently, metal complex solubilities and metal extraction using chelating agents have been investigated using a stainless steel SFE system. Metal complexes are usually soluble in supercritical carbon dioxide (SC-CO2) and so this was the fluid of choice for the experiments. During the chelation step, temperature and pressure have great effect on the extraction efficiency. Using a metal salt as a sample, and hexafluoroacetylacetone (HFAcAc) as a ligand, the most efficient extraction conditions were found to be 60°C and 400atm. However, when contaminated soil was used as a sample, extraction efficiency fell. This was thought to be related to the corrosion of the stainless steel SFE system by the ligand HFAcAc. During the 30min extraction period, 1678μg of iron was obtained. The corrosion of the SFE system was much greater during the soil extraction than during both the metal salt extraction and metal complex solubility experiments. Copyright (C) 2000 Elsevier Science B.V.

Objective To review the present position of the western swamp tortoise (Pseudemydura umbrina) as an endangered species and significant health issues affecting efforts to save it from extinction. Procedure A retrospective analysis of the husbandry, hospital and pathology records of the western swamp tortoise captive breeding program at Perth Zoo. Results In 1987 a captive breeding project was developed to prevent the extinction of the western swamp tortoise but an outbreak of a necrotising dermatitis in 1989 threatened the survival of the captive bred hatchlings. Less severe outbreaks occurred in 1990 and 1993, with isolated cases in between. Of 283 tortoises that were born in captivity or came into captivity from the wild, 37 (13.1%) were affected, comprising 37% of all males, 26% of all females and 13% of animals of unknown gender. Of the affected animals, 70% were less than 2 years of age and 29% were older. Males were 1.6 times more likely to be infected than females but this difference was not statistically significant (P = 0.27). Culture of the lesions consistently yielded unidentified Pseudomonas sp. Conclusion Improved husbandry, such as strict maintenance of water quality and temperature conditions similar to that of the animal's natural habitat, and monitoring the health of individual tortoises have successfully controlled skin disease in the captive breeding of the western swamp tortoise.

The saying ‘time is money’ is becoming ever more common in the laboratory environment. There has always been a need to control analytical costs in terms of solvent usage and disposal, consumable expenditure and administration overheads. However, there is currently an even greater emphasis on the time an analysis takes, and this has led to the development of automated commercial extraction methods using solvent, microwave-assisted and supercritical fluid extraction (SFE) systems. To automate the analytical process fully the extraction and analysis procedures need to be combined to produce a coupled on-line technique that minimises the sample handling and preparation steps which contribute to analyte loss, degradation and/or contamination. This direct transfer of extracted analytes into the analyte separation and detection system produces a more sensitive analysis and increases the laboratory sample throughput. For example, the extraction and chromatographic analysis of real-world samples with low (ppm—ppb) analyte concentrations can be achieved within 1 h using an on-line approach [1-3].

Sample preparation is often proved the most time consuming part of a chromatographic assay because it usually involves an extraction procedure with organic solvents and the subsequent need to evaporate the solvent to concentrate the extract.

A model for dynamic supercritical fluid extraction (SFE) is proposed, which contains the steps of: rapid fluid entry into the matrix; a reversible release process such as desorption from matrix sites or penetration of a biological membrane; transport, by diffusion or otherwise to the edge of a matrix particle; and removal by biological membrane; transport, by diffusion or otherwise to the edge of matrix particle; and removal by solvation in the fluid. It can explain the following kinetic features of dynamic SFE : (i) a slow initial extraction; (ii) a more rapid extraction phase; and (iii) a slow final phase, which can be so slow that it appears that extraction is complete when only a fraction of the solute present in the matrix has been recovered. The model may be used to extract values for the parameters quantifying the individual steps, to understand further the SFE process.

Human macrophage-like accessory cells were analyzed as they emerge in the absence of extrinsic antigens during fetal development. Monoclonal antibodies to monocytes/macrophages were used in combination with antibodies to HLA class II molecules. In the yolk sac and mesenchyme sampled at wk 4 to 6 of fertilization age, cells with dendritic morphology formed two populations distinguishable by phenotypic criteria: type i (majority) carried both macrophage-associated (RFD7+) and monocyte-associated markers (UCHM1+) but no detectable HLA-DR antigen, and type ii (minority) constitutively expressed class II (HLA-DR and -DP) but no RFD7 and UCHM1. The emergence of this heterogeneity preceded the formation of both thymus and bone marrow. During additional development, type i and type ii cells seeded to different microenvironments and underwent some additional phenotypic changes. Cells of type i, the RFD7+ population with high lysosomal (acid phosphatase) activity, were seen in the thymic cortex, marginal zone of lymph nodes, splenic red pulp, and in the midst of erythropoietic activity within the bone marrow. These cells were UCHM1- and class II-. Cells of type ii formed the population of HLA-DR+, RFD7- interdigitating cells, early inhabitants of T cell areas in the developing thymic medulla, lymph nodes, spleen, and tonsil. The Type ii cells that had already settled in their nichès expressed not only HLA-DR and -DP but also HLA-DQ, and another class II antigen identified by the antibody RFD1, which shows the restricted tissue distribution of HLA-DQ, but is governed by genes that are outside of and telomeric to the HLA-DQ region (or HLA-DR). Finally, subpopulations of macrophages (RFD7+, acid phosphatase-positive) in the fetal gastrointestinal and hepatic systems were HLA-DR+; the latter appear to include precursors of Kupffer cells in the developing liver.

he ontogeny of cells containing the enzyme terminal deoxynucleotidyl transferase (TdT) in human fetal liver, bone marrow, and thymus has been studied using a highly specific antiserum to TdT together with monoclonal antiprecursor cell antibodies in double and triple marker immunofluorescence. TdT+ cells were first observed in fetal liver at 12 wk of gestation and accounted for 55% of the lymphoid-like cells isolated after Ficoll-Hypaque separation. TdT+ cells were first observed in the bone marrow 16 wk after gestation. Like TdT+ cells in normal infant bone marrow, the majority of TdT+ cells in fetal liver and bone marrow expressed both BA-1 and RFB-1 antigens. This suggests that fetal TdT+ cells include progenitors of the B lineage (BA-1+) and perhaps of thymocytes (RFB-1+). Nevertheless, TdT was not observed in fetal thymocytes until after 20 wk of gestation, although thymic blasts and the majority of thymocytes were strongly RFB-1+ from 12 wk of gestation. These results clearly show that fetal thymus is first populated by TdT, RFB-1+, BA-1 cells, but does not exclude the fact that a second “wave” of TdT+ prothymocytes, possibly bone marrow derived, also exists.

In man, during fetal development the B cell populations show distinct phenotypes at different tissue sites. The pre-B and B lymphocytes of the fetal liver and bone marrow express IgM and B cell markers, B1 (CD20) and BA-1 (CD24). These "early" cells are negative with a number of other reagents, anti-IgD, RFB4 (CD22), RFB6 (CD21), and RFA-2, which on the other hand recognize peripheral B cells. These peripheral B lymphocytes in the developing fetus are heterogeneous. The diffusely distributed B cells in the earliest lymph node samples, 16 to 17 wk of gestational age, and from 16 to 21 wk in the spleen, are strongly IgM+ (IgD+,RFB4+,RFB6+, and RFA-2+) but lack T cell-associated markers such as T1 (CD5, p 67,000 dalton equivalent of murine Ly-1) and Tü-33. In fetal lymph nodes, primary nodules develop around the follicular dendritic (FD) cells from 17 wk onward, and contain a virtually pure population of B cells; B1+,BA1+,RFB4+,RFB6+,RFA-2+, which simultaneously express IgM,IgD together with T1 (CD5), a T cell-associated antigen. A sizeable subpopulation of these IgM+,T1+ cells are also positive for Tü-33, another T cell-associated marker. In the spleen, the B cells of the IgM+,IgD+,T1+ type appear in smaller numbers and only relatively late around wk 22. These cells are diffusely distributed at first, and start accumulating around the small FD cell clusters as soon as these emerge about the 23rd gestational wk. At that time, the IgM+,T1+B cells can also be washed out from the peritoneal and pleural cavities. The T1+,IgM+B cells may represent the normal equivalent cells of B chronic lymphoid leukemia and centrocytic lymphoma, and appear to be the counterpart of Ly-1+,IgM+B cells in the mouse.