PubMed Health⌕ Search

Biomedical subjects

S A Bentley

Publications and source records attributed to S A Bentley.

At least 19 recordsLinked to original sources
PubMed1991

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies.

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.

Animals↗
PubMed1990

Neutrophil myeloperoxidase measurement uncovers masked megaloblastic anemia.

We report the observation of a high neutrophil myeloperoxidase activity (MPXI) in patients with megaloblastic anemia. MPXI is rapidly measured as part of an automated complete blood count (Technicon H*1, Technicon Instruments Corp, Tarrytown NY). We describe the range of MPXI levels in healthy and patient populations and in 10 cases of megaloblastic anemia, including five having elevated mean cell volume (MCV) and five without macrocytosis. Regardless of the MCV, our megaloblastic patients had hypersegmented neutrophils and elevated MPXI levels without visible alteration of granule content. MPXI measurement may be particularly useful in identifying cases of "masked megaloblastic anemia" where the MCV is below 100 fL. The advantage of the MPXI over other methods of uncovering masked megaloblastic anemia is its simplicity when performed as part of a routine complete blood count on an automated hematology instrument.

Adolescent↗
PubMed1990

Biochemical and functional characterization of proteoglycans produced by Sl/Sld murine bone marrow stromal cell lines.

The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (Sl/Sld) animals and two control stromal cell lines, one (+/+2.4) from a non-anemic littermate, and one (GBl/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCl density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co-culture of an IL-3-dependent cell line (FDC-P1) with cell-free ECM preparations from an Sl/Sld and a control (GBl/6) stromal cell line, with and without pre-incubation with IL-3. Cell-free ECM preparations from Sl/Sld and control cell lines supported FDC-P1 growth to an approximately equal extent after pre-incubation with IL-3. FDC-P1 growth support by ECM preparations from both cell lines was also observed without IL-3 pre-incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.

Anemia, Macrocytic↗
PubMed1990

Automated differential white cell counts: a critical appraisal.

The differential leukocyte count represents a substantial proportion of the workload in routine haematology laboratories. The traditional procedure, in which a technologist scans a Romanowsky-stained blood film and classifies 100 cells, is both labour-intensive and imprecise. The imprecision of the visual DLC is primarily a function of the small number of cells counted. To achieve a significant improvement, however, it would be necessary to count an enormous number of cells, which would be extremely costly in terms of human time and effort. Automation is thus desirable for both economic and clinical reasons. Of the various technological approaches proposed, only two have been developed to the level of marketable devices: digital image processing, and flow cytochemistry. In digital image processing systems, Romanowsky-stained blood films were scanned by a computer-controlled microscope, and leukocytes identified on the basis of parameters analogous to those used by human observers. These systems were relatively slow, offering only a limited degree of automation, and therefore failed to provide significant improvements in terms of workflow and counting statistics. No such instruments are currently marketed. Flow-cytochemical analysers, which classify leukocytes primarily on the basis of size and myeloperoxidase activity, have been available commercially for 15 years, and their clinical utility is well established. A number of haematology analysers offer screening DLCs, providing neutrophil and lymphocyte counts in cases that do not have major abnormalities of the leukocyte population. The performance of these systems depends in large part on their operating environment. Three new automated DLC analysers employing flow cytometric technology have recently been announced, but these have not yet been formally evaluated.

Autoanalysis↗
PubMed1990

Quality control and the differential leukocyte count.

The differential leukocyte count (DLC) has been an important semi-quantitative method for describing the leukocyte population as visualized on examination of the peripheral blood film. Evaluation of the DLC was never a diagnostic procedure in its own right, nor was it the primary purpose of the blood film evaluation. It is the most time-consuming element of the blood film examination, and therefore a primary candidate for automation, which in turn has led to an artificial separation of DLC enumeration from blood film evaluation. Nevertheless, it is being used as a quality assurance test for automated DLC procedures. While this may divert valuable effort away from the central function of the diagnostic haematology laboratory, namely haematological diagnosis, it has become a quality assurance requirement. Quality is best assured when haematological case data, including history, cell counts, indices, and DLC data, are evaluated by appropriately trained experts before being reported to clinicians, with examination of the blood film as indicated.

Automation↗
PubMed1989

Biosynthesis of proteochondroitin sulfate by HL-60 human promyelocytic cells.

Human promyelocytic cells (HL-60) were labeled with 35S-sulfate and either 3H-glucosamine or 3H-serine as precursors. Accumulation of 35S-labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/10(5) viable cells reached a plateau level after 24 h. Virtually none of the cell-associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE-Sephacel chromatography, isopyknic CsCl gradient centrifugation, and gel filtration chromatography. HL-60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL-4B and Kav = 0.32 on Sepharose CL-6B, recovered primarily from the high-density fractions of a dissociative CsCl gradient (rho greater than 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4-sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL-60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S-sulfate incorporation to 45% of control values or 32%, expressed as activity/10(5) cells. Proteoglycans synthesized by DMSO-treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.

Aggrecans↗
PubMed1989

The clinical utility of discriminant functions for the differential diagnosis of microcytic anemias.

Several groups of authors have derived discriminant functions (DFs) based on red cell indices (primarily MCH, MCV, and RDW) that can be used to differentiate iron deficiency from thalassemia minor. The Technicon H*1 analyzer provides a direct MCHC measurement (termed the CHCM), in addition to the conventional computed value (Hgb/PCV). To evaluate the clinical utility of red cell discriminant analysis, chart review was performed in 176 cases for which hemoglobin characterization and quantitation studies had been requested. Six published discriminants were evaluated for cases of clearly defined iron deficiency anemia and thalassemia minor. Overall diagnostic efficiency ranged from 50%-82%, and the diagnostic performance of three of the discriminants failed to achieve statistical significance. Mean values for both MCHC and CHCM were significantly lower in patients with iron deficiency than in patients with other causes of microcytic anemia. It was also observed that MCHC was significantly greater than CHCM in patients with iron deficiency anemia, but not in patients with other causes of microcytic anemia. Both MCHC and the difference between MCHC and CHCM showed potential value as parameters for the differential diagnosis of iron deficiency from other causes of microcytic anemia. It was noted, however, that in 67% of the cases studied, the use of a DF could not have resolved the diagnosis to the extent that hemoglobin characterization and quantitation studies were no longer indicated.

Anemia, Hypochromic↗
PubMed1988

Bone marrow stromal proteoglycan heterogeneity: phenotypic variability between cell lines and the effects of glucocorticoid.

Hematopoiesis in vivo is dependent upon the interaction of hematopoietic stem cells with a complex microenvironment, of which stromal proteoglycans are an important functional component. Certain bone marrow stromal cell lines provide a microenvironment that supports hematopoiesis in vitro, a function that is dependent upon glucocorticoid supplementation. Proteoglycan synthesis in the hematopoietic-supportive D2XRII, Bl6 and 14F1 bone marrow stromal cell lines was studied by 35S-sulfate precursor labelling and ion-exchange separation, followed by isopyknic CsCl density centrifugation and gel filtration HPLC. The effects of glucocorticoid were also investigated. A similar pattern of proteoglycan heterogeneity was observed in all three cell lines, although there was considerable quantitative variation. All cultures synthesized three species of chondroitin/dermatan sulfate (CS/DS) proteoglycans: DS1, excluded from a Bio-Sil TSK-400 HPLC column, and DS2, eluting at Kd = 0.31, were present mainly in the culture media. The smallest (DS3) eluted at Kd = 0.63 and was present mainly in the cell layers. CS/DS species were the major proteoglycans in all cultures. Hydrocortisone-free cultures also synthesized heparan sulfate (HS) proteoglycans, including a cell-associated form (HS1), partially excluded from the TSK-400 column, and a secretory form (HS2), eluting at Kd = 0.15. D2XRII cells also secreted an apparently-unique, high-density proteoglycan, Kd = 0.65, into the culture medium. Hydrocortisone at 10(-6) M virtually abolished HS proteoglycan synthesis in all three cell lines, and altered the pattern of CS/DS proteoglycans in the culture media, increasing the quantity of DS1 and DS3, and reducing the quantity of DS2.

Animals↗
PubMed1988

Stability of hematologic parameters in healthy subjects. Intraindividual versus interindividual variation.

An automated complete blood count with white blood cell differential was performed yearly on successive groups of healthy second-year medical students from 1979 through 1987. For three classes (1984-1987), the counts were repeated on the same people nine months later. These data demonstrated that the mean value of all hematologic parameters was quite stable over nine years. This allowed for an estimation of the upper limit for the combined effects of drift in accuracy, precision, and biologic stability. The stability was achievable despite an evolution in technology and quality assurance methods over that period. A comparison of intraindividual versus interindividual variation demonstrated that a normal range based on population statistics may be less sensitive than a normal range established for a person during routine health maintenance, especially for the platelet count.

Adult↗
PubMed1987

Diagnosis of infective and inflammatory disorders by flow cytometric analysis of blood neutrophils.

The utility of neutrophil parameters provided by two flow cytometric hematologic analyzers (the H-1 and H6000, Technicon Instruments Corporation, Tarrytown, NY) was investigated for the diagnosis of infective and/or inflammatory disorders. The test population of 156 hospital patients was selected on the basis of a blood culture request. Positivity or negativity for infective and/or inflammatory disease was inferred from chart review. The parameters evaluated included the absolute neutrophil count, the lobularity index, and the left shift flag from the H-1, the percentage of high peroxidase cells from the H6000, the routine laboratory band count, and a reference band count. Significant intercorrelations were observed between these parameters. The diagnostic performance of the routine laboratory band count was significantly inferior to that of all other parameters. At equivalent points on their receiver operating characteristic curves, the diagnostic efficiencies of the remaining tests ranged from 61.5% for the lobularity index to 67% for the left shift flag and the percentage of high peroxidase cells. These differences were not significant statistically.

Flow Cytometry↗
PubMed1987

Interpretation of red cell survival data for in vivo compatibility testing: a normal value study.

Short-term survival studies are sometimes required to determine the compatibility of donor red cells. The results of these studies are generally expressed as per cent survival at 60 min. The present study was undertaken to investigate the potential for more sophisticated data analytical techniques to improve sensitivity. In one group of eight healthy male volunteers, autologous red cells were labelled with 51chromium and injected immediately, while in a second group, red cells were stored for 5 d prior to injection. In both groups, eight to 10 samples were collected in the first 6 h and another 10-12 samples over the next 4 weeks. Estimation of the 60 min per cent survival was insufficiently sensitive to detect 'physiological' haemolysis following injection of 5-d-old autologous blood. Regression analysis of 6 h survival data, however, demonstrated significantly higher red cell clearance rates in these cases than in those receiving fresh cells, with a mean 24 h loss of 3.3% of activity. The upper limit for the 6 h red cell clearance rate was 1.63%/h after fresh autologous blood and 2.43%/h after 5-d-old blood. The significance of these findings is discussed and a protocol suggested for the analysis of short-term red cell survival data.

Adult↗
PubMed1987

Proteoglycan synthesis in two murine bone marrow stromal cell lines.

There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3-2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3-2A does not. Cells were labeled with 35S-sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3-2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3-2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.

Animals↗
PubMed1986

Evaluation of an automated hematology system (Technicon H-1).

We evaluated the Technicon H-1 hematology system, a new third-generation blood cell analyzer that performs a complete blood cell count, including red blood cell (RBC) morphometric studies and a full white blood cell differential count. Linearity and precision were acceptable over the entire clinical range. We compared the H-1 with the Technicon H-6000 and the Coulter S Plus IV. Close correlation was observed for all equivalent parameters except the mean corpuscular hemoglobin concentration (MCHC), the mean platelet volume, and the basophil count. Poor correlation for the MCHC reflects the novel methodology of the H-1 for measuring RBC size and, independently, RBC hemoglobin content. The H-1 MCHC correlates with hypochromia as assessed morphologically, while the MCHC of the H-6000 or S Plus IV does not. Further technological innovations include measurement of the mean neutrophil peroxidase content and nuclear lobularity.

Blood Cell Count↗
PubMed1979

Bone marrow fibre production in myelofibrosis: a quantitative study.

A digital image processing technique has been used to quantitate bone marrow fibre in histological material (both biopsy and necropsy) from 17 cases of myelofibrosis. An attempt was made to correlate the results of these measurements with other diagnostically relevant parameters. Bone marrow fibre was found to correlate significantly with absolute neutrophil count, the absolute count of immature granulocytic cells and with the number of immature granulocytic cells expressed as a percentage of the total leucocyte count. There was no significant correlation between marrow fibre content and platelet count for the series as a whole, but for those cases in which measurements were made following splenectomy these parameters were highly correlated. Bone marrow fibre content did not appear to correlate with liver or spleen weight at necropsy, nor with the duration of clinical manifestations of the disease. The significance of these findings is discussed.

Bone Marrow↗
PubMed1978

Staining properties and stability of a standardised Romanowsky stain.

An evaluation of the standardised Romanowsky stain of Marshall et al. has been made in a routine haematology laboratory. It was noted that this stain had several advantages over the May-Grünwald Giemsa stain used in most British laboratories. These advantages include ease and speed of preparation, a shorter staining time, and reproducibility of results. These results are described in detail. The stability of the stock stain solution and of the 'working' stain (stock + buffer) has been studied by, respectively, thin-layer chromatography and visible spectroscopy. No change was detected in the composition of the stock solution at ambient temperature over a period of six months. Stability was unaffected by the composition of the container (polyethylene, PyrexTM, or soda-glass) or by daylight. The 'working' solution was stable for 3 hours. Thereafter a precipitate is formed, consisting of thiazine dyes and eosin in a molar ratio of approximately 2:1.

Chromatography, Thin Layer↗
PubMed1978

Standardization of Romanowsky stains. The relationship between stain composition and performance.

A panel of 17 eminent haematologists has assessed the performance of 5 Romanowsky stains prepared from pure component dyes, comparing the suitability and acceptability of these stains for the preparation of routine blood and bone-marrow films. It was found that the results obtained using the stain described by Marshall et al (1975) were comparable to those obtained using a modification of the stain described by Wittekind et al (1976). The performance of the 3 other stains was less acceptable. Variations in stain formulation have been correlated with stain performance.

Blood Cells↗