Optimisation and validation of a sensitive high-performance liquid chromatography assay for routine measurement of pyridoxal 5-phosphate in human plasma and red cells using pre-column semicarbazide derivatisation

There are few studies in which direct measurement of vitamin B6 status in both plasma and red cells has been assessed. The aims of the present study were to evaluate the use of a simple, robust HPLC method of direct pyridoxal 5'-phosphate (PLP) measurement in plasma and red cells and to assess its use in establishing reference ranges in a healthy population. A reverse phase HPLC method with pre-column derivatisation using semicarbazide for the simultaneous measurement of PLP, its degradation product, 4-pyridoxic acid (PA) and pyridoxal (PL) in plasma and red cells was developed. Pre-column derivatisation, reverse phase chromatography and detection procedures were optimised. The recovery, precision, linearity and sensitivity of the assay for plasma and red cell PLP, PA and PL was established. The recovery of PLP was greater than 95% for both plasma and red cell samples. The Intra and Inter batch imprecision for PLP was less than 6% and 7%, respectively. The method for PLP was linear up to at least 1000 nmol/l and the detection limit was 2.1 nmol/l (limit of quantification; 5.8 nmol/l). Accuracy of PLP measurements in plasma were acceptable, showing a mean bias of 4.5% from the mean value of laboratories (N=34) participating in an external quality assurance scheme. Geometric mean (95% reference intervals) for plasma and red cell PLP in the healthy subjects (N=126) were 56 (21-138) nmol/l and 410 (250-680) pmol/g Hb, respectively. There was a strong positive correlation (r(2)=0.81) between plasma and red cell PLP levels in the reference population. The HPLC method described was found to be suitable for the routine measurement of PLP in both plasma and red cells.     

An automated method of differential red blood cell classification with application to the diagnosis of anemia.

A method of automated red cell analysis suitable for the rapid classification of large numbers of red cells from individual blood specimens has been developed, and preliminarily tested on normal bloods and clinically proven cases of anemias and red cell disorders. According to this method digital image processing techniques provide several features relating to shape and internal central pallor configurations of red cells. These features are used with a fully automated decision logic to rapidly provide a quantitative "red cell differential" analysis, a report of the percentage subpopulations of recognized categories of red cells. For each subpopulation, measurements of mean cell area, mean cell hemoglobin content and mean cell hemoglobin density are provided. The nine types of red cell disorders studied with this method were: (a) iron deficiency anemia, (b) the anemia of chronic disease, (c) beta-thalassemia trait, (d) sickle cell anemia, (e) hemoglobin C disease, (f) intravascular hemolysis, (g) hereditary elliptocytosis, (h) hereditary spherocytosis, and (i) megaloblastic anemia due to folic acid deficiency. Preliminary indications are that the red cell differential is useful in distinguishing between these conditions.     

基于GIS识别生态保护红线边界的方法——以北京市昌平区为例

生态保护红线是中国生态保护的一项重要创新,已被列为国家监管生态环境的重要政策。国家环境保护部于2015年发布了《生态保护红线划定技术指南》,确定了生态保护红线的划定方法,但是在识别生态保护红线边界时还存在一些技术难点。针对这一问题,本文探索了生态保护红线边界的优化方法。提出了生态保护红线边界优化遵循基本原则。以北京市昌平区为例,阐明了如何利用Arc GIS软件将划定生态保护红线的技术流程,包括评价各类生态功能区和敏感区,识别生态保护红线范围,准确定位生态红线边界3个主要步骤。最后通过对比优化前后的生态保护红线划定方案,阐明了该方法的可行性和存在的问题。     

大流行流感疫苗血清学检测中马血球和鸡血球血凝抑制试验方法的比较

血凝抑制试验(HI)是评价季节性流感疫苗免疫效果的经典方法。由于对人、禽流感病毒的受体不同,不同来源的红血球检测敏感性可能有差异。实验中比较了经典的鸡血球HI方法和国外报道的马血球HI方法,发现两种方法在检测大流行流感疫苗接种者血清时,于不同毒株抗原检测时表现各不相同,检测结果差异在2倍之内,说明经典的鸡血球HI方法仍适用于评价大流行流感疫苗的免疫效果。     

圈养小熊猫几种病毒的PCR检测

本文采用巢式PCR/ RT-PCR 方法,对我国10 个动物园中无临床症状的圈养小熊猫的71 个肛拭子和61 个唾液拭子样品,进行犬瘟热病毒(CDV)、犬细小病毒(CPV)、犬冠状病毒(CCV)、犬腺病毒(CAV)和犬疱疹病毒(CHV)的检测,以评估我国圈养小熊猫是否面临这几种病毒的威胁。对阳性PCR 结果进行测序分析,并与GenBank 上的序列进行比较。结果,在肛拭子样品中检测到3 个CPV 和6 个CCV 阳性结果,经测序后,与GenBank 上序列的同源性分别达99% 和100% 。而在唾液拭子样品中没有检测到任何阳性结果,且CDV、CAV和CCV 的检测结果均为阴性。从阳性CPV 的肛拭子样品中分离到一株细小病毒毒株,表明圈养小熊猫已受到细小病毒和犬冠状病毒的感染,今后应加强这两种病毒的预防工作。本文所采用的PCR 方法检测病毒性疾病,能检测到微量的病毒模板,可对小熊猫病毒性感染进行早期诊断。     

Detection of cellulolytic fungi by using Congo red as an indicator: a comparative study with the dinitrosalicyclic acid reagent method

Cellulolytic fungi can easily be screened within 2 d for the production of cellulolytic enzymes by staining with Congo red, or by measuring the amount of reducing sugar (glucose) produced with the dinitrosalicyclic acid reagent method. Endoglucanase activity is visible in carboxymethyl cellulose agar plates after staining with Congo red, and fixing with HCL or NaOH. This method is essentially based on the interaction of Congo red with intact β-(1-4)-D-glucans in carboxymethyl cellulose. Endoglucanase and exoglucanase activities of cellulases are quantitatively measured with the dinitrosalicyclic acid reagent method. The enzymatic activity detected with Congo red is compared with that obtained by the dinitrosalicyclic acid reagent method.     

A scene-segmentation method for multispectral cell images

A scene-segmentation method for two-color digitized images acquired from a Papanicolaou-stained cervical smear is proposed. The method first segments a scene into background, red cytoplasm, blue cytoplasm and nuclear regions by a pixel-wise classification and then merges the segmented regions for both types of cytoplasm into a single region. To create the minimum-distance classifier used for the pixel classification, class median vectors are selected from a two-dimensional histogram formed from the optical densities in the red and green images (scanned at 610 nm and 535 nm, respectively). Reference points defined from knowledge about the two-color images played an important role in selecting the vectors for the red and blue cytoplasm. This method was applied to 33 sets of the two-color images. The resulting segmented regions corresponded well with regions apparent to the the human observer. Three different investigations related to the method were carried out; these studies confirmed the suitability of the proposed method.     

Association of phosphofructokinase and aldolase with the membrane of the intact erythrocyte

The binding of phosphofructokinase and aldolase to the membrane of the intact human erythrocyte was assessed by the rapid hemolysis/filtration method of Kliman and Steck (Kliman, H. J., and Steck, T. L. (1980) J. Biol. Chem. 255, 6314-6321). We found that about 50% of the phosphofructokinase was membrane-bound in fresh red cells prior to hemolysis. Binding was not significantly altered by deoxygenation. Approximately 40% of aldolase was membrane-associated in fresh red cells. In outdated, blood-banked red cells, aldolase was 73% membrane-bound while, following metabolic repletion, 40% of the enzyme was membrane-associated. These results support the hypothesis that certain glycolytic enzymes in the red cell are membrane-bound in a rapidly reversible and metabolically sensitive fashion.     

Morphology of hair pigmentation in wild red foxes,silver foxes,and their hybrids

The effects of dominant allele Ar of locus Agouti on the morphology of hair pigmentation were described in foxes. The Ar allele was shown to determine the type of melanin and its content in hair with no effect on the morphology of pigment granules and their distribution throughout a hair. Using the method of electron spin resonance (ESR), the types of melanin (eumelanin and pheomelanin) and their content in the hair of red (ArArEE) and silver (aaEE) foxes and their hybrids (AraEE) were determined. In silver foxes, only one type of melanin (eumelanin) was found. In red foxes and their hybrids (which are phenotypically similar but darker than red foxes), both types of melanin (eu- and pheomelanin) were found. The highest melanin content was detected in the coat of silver foxes. In the hybrids, the total melanin content was lower than in silver foxes, but significantly higher than in red foxes. In red foxes, the contribution of pheomelanin to the total hair melanin content was twice as large as in the hybrids.     

A simple method for determination of red blood cell mechanical fragility in the rat

A method for measuring the mechanical fragility of red blood cells suitable for use in small laboratory animals, such as rats, is reported because of lack of such data in the literature. Whole blood is mixed with phosphate buffered saline in a tube containing glass beads. The tubes are rocked for 90 minutes, centrifuged and the percent hemolysis determined. Varying the osmolality of the saline suspending medium had little effect on the mechanical fragility of rat red cells prior to the NaCl concentrations at which a significant change in osmotic hemolysis occurred. The duration of rocking increased the mechanical fragility. Varying the pH (6.4-8.0) had no effect. The size of the glass beads changed the mechanical fragility as did varying temperature. The mean mechanical fragility of rat red blood cells was 46% hemolysis (80 adult male animals). Because of the small volume of blood required with this method, mechanical fragility of red cells of other small laboratory animals also may be determined.     

An optical method for the determination of platelet count in platelet samples contaminated with red blood cells.

We present a simple photometric method to determine the total concentration of platelets present in a sample independently of red blood cell concentration. Standard optical density curves for platelet samples ranging in concentration from 0 to 1.5 x 10(9) cells/ml and contaminated with red blood cells ranging in concentration from 0 to 0.03 x 10(9) cells/ml are determined. A study of the absorbance spectra of red blood cells and platelets suggests that by calculating the absorbance difference between two wavelengths, an estimate of red blood cell concentration can be made. Then, in the second step of this two-step method, the individual absorbance measurements at the two wavelengths are matched to the standard values determined previously to derive an estimate of platelet concentration. In a trial of 62 unknown platelet samples contaminated with red blood cells, the standard deviation for the error in platelet count was 0.16 x 10(9) cells/ml with a mean difference of 0.011 x 10(9) platelets/ml. We conclude that our method may be useful in laboratories not equipped with electronic cell counters as well as in applications such as the development of noninvasive measurements of platelet concentration in platelet transfusion packs.     

Detection of surface differences between two closely related cell populations by partitioning. Erythrocytes from inbred and out-bred rats and rat strains

We have recently developed a new and powerful method capable of detecting, by purely physical means, surface differences between closely related red (or other) cell populations. The procedure consists of isotopically labeling (with [⁵¹Cr]chromate) aliquots of red blood cell populations. Such labeled cells are mixed with an excess of unlabeled red cells to which they are to be compared. The mixtures are subjected to countercurrent distribution in either a charge-sensitive or a non-charge-sensitive dextran-poly(ethylene glycol) aqueous phase system. The distribution curves are analyzed for total cells (in terms of hemoglobin absorbance) and labeled cells (in terms of cpm). Changes in the relative specific activities through the distribution curves are indicative of subtle differences in surface properties between such cell populations. Using this method we have found that erythrocytes from arbitrarily chosen (presumably hematologically normal) individuals differ. In the current work we have examined the surface properties of erythrocytes from Sprague-Dawley and from Lewis rats. This was done with a view to determining whether (a) differences of the type found between different humans can also be detected in other species and (b), if such differences do exist, to examine, by study of the highly inbred Lewis rat strain, whether the differences appear to have a genetic or an acquired basis. It was found that the surface properties of erythrocytes from Lewis and Sprague-Dawley rats differ as do erythrocytes among rats of the Sprague-Dawley strain. No difference was found between red blood cells from different rats of the inbred Lewis strain. These results indicate that the surface differences between red blood cells from different rats detected by partitioning have a genetic rather than acquired origin.     

柑橘全爪螨种群空间格局的地学统计学分析

应用地学统计学方法分析了柑橘园主要害螨柑橘全爪螨Panonychus citri（McGregor）种群的空间格局及其动态。结果表明,柑橘全爪螨种群具有空间相关性,变程介于1．10～21．0m,其半变异函数主要符合高斯模型,表现为聚集分布,其中3月、8月和9月的聚集强度较大;种群空间格局动态显示,4月、10月为该种群的两个发生高峰期,柑橘全爪螨种群数量快速上升扩散。地学统计学方法能够应用于柑橘全爪螨种群的空间格局分析,并有助于对该害螨进行发生预测与控制处理。     

A dual fluorescence flow cytometric analysis of bacterial adherence to mammalian host cells

Flow cytometry has provided a powerful tool for analyzing bacteria-host cell associations. Established approaches have used bacteria, labeled either directly with fluorochromes or indirectly with fluorescently conjugated antibodies, to detect these associations. Although useful, these techniques are consistently unable to include all host cells in the analysis while excluding free, aggregated bacteria. This study describes a new flow cytometry method of assessing bacterial adherence to host cells based on direct fluorescent labeling of both bacteria and host cells. Eukaryotic host cells were labeled with PKH-26, a red fluorescent dye, and bacteria were labeled with fluorescein isothiocyanate, a green fluorescent dye. The red host cells were gated and the mean green fluorescence intensity (MFI) of these red cells was determined. We used MFI values obtained from control samples (unlabeled and labeled host cells with unlabeled bacteria) to eliminate contributions due to autofluorescence. The final MFI values represent fluorescence of host cells resulting from the adherent bacteria. Because all red fluorescent cells are analyzed, this method includes all the eukaryotic cells for analysis but excludes all free or aggregated bacteria that are not bound to target cells.     

Rapid assessment of islet viability with acridine orange and propidium iodide

Summary A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential. Preliminary reports of this work were presented at two meetings and were published in abstract form (24,25). This research was supported in part by the National Institutes of Health, Bethesda, MD, grant DK 18115.     

Genetic validation and characterization of RAPD markers differentiating black and red spruces: molecular certification of spruce trees and hybrids

 Picea mariana (black spruce) and P. rubens (red spruce) are closely related species which are difficult to differentiate morphologically. RAPD markers differentiating black and red spruces have been previously identified. In the present study, genetic validity of these markers was determined using samples representing range–wide provenances. Their applicability for certifying genetic identity of individual black, red trees and their hybrids from several sympatric and allopatric locations was demonstrated. These diagnostic fragments of both red and black spruce were present at a frequency of over 0.95 in allopatric provenances, but at a lower frequency in some sympatric provenances (0.43–1.00). Natural populations of red spruce exhibiting typical red spruce phenotype contained black spruce diagnostic RAPD fragments and black spruces growing in bogs with typical bog black spruce morphology, contained red spruce-specific RAPD markers. Some major RAPD markers were cloned and sequenced. The results reveal an extremely high degree of identity between the random primer and the primer binding sites on the genome. Amplification of black and red spruce genomic DNA with designed primers flanking the species-diagnostic RAPD markers indicates that most of RAPD markers used to differentiate black spruce from red spruce are not species specific since these sequences were detected in several spruce species using a more sensitive detection method. Received June 17, 2002; accepted August 5, 2002 Published online: February 4, 2003     

Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.     

Radiosensitivity of early and late M-phase HeLa cells isolated by a combination of fluorescent ubiquitination-based cell cycle indicator (Fucci) and mitotic shake-off

The mitotic shake-off method revealed the remarkable variation of radiosensitivity of HeLa cells during the cell cycle: M phase shows the greatest radiosensitivity and late S phase the greatest radioresistance. This method harvests all M-phase cells with a round shape, making it impossible to further subdivide M-phase cells. Recently, the fluorescent ubiquitination-based cell cycle indicator (Fucci) was developed; this system basically causes cells in G(1) to emit red fluorescence and other cells to emit green fluorescence. Because the green fluorescence rapidly disappears at late M phase, two-dimensional flow cytometry analysis can usually detect a green(high)/red(low) fraction including S-, G(2)- and early M-phase cells but not a transitional fraction between green(high)/red(low) and green(low)/red(low) including late M-phase cells. However, combining the shake-off method concentrated the transitional fraction, which enabled us to separate early and late M-phase cells without using any drugs. Here we demonstrate for the first time that cells in early M phase are more radiosensitive than those in late M phase, implying that early M phase is the most radiosensitive sub-phase during the cell cycle.     

一种胆固醇氧化酶高产菌株筛选方法的建立

用亚硝基胍(1 mg/mL)与超声波(200 W, 50 kHz)复合诱变的方法, 对产胆固醇氧化酶短杆菌Brevibacterium sp. DGCDC-82进行诱变处理, 得到一株桔红色突变株其产胆固醇氧化酶能力提高140%, 酶活达到1.24 U/mL, 又用同样的方法对桔红色突变株进行回复突变处理, 得到一株白色回复突变株和一株淡粉色回复突变株, 两株回复突变株产胆固醇氧化酶的能力又明显下降, 酶活分别为0.17 U/mL和0.69 U/mL。说明短杆菌Brevibacterium sp.产胆固醇氧化酶能力与其产红色素成正相关偶联关系, 这种相关性模型的建立可以作为以后诱变或定向进化研究的筛子。     

一种胆固醇氧化酶高产菌株筛选方法的建立

用亚硝基胍(1 mg.mL)与超声波(200 W,50 kHz)复合诱变的方法,对产胆固醇氧化酶短杆菌 Brevibacterium sp.DGCDC-82 进行诱变处理.得到一株桔红色突变株其产胆固醇氧化酶能力提高140%,酶活达到1.24 U/mL,又用同样的方法对桔红色突变株进行回复突变处理,得到一株白色回复突变株和一株淡粉色回复突变株,两株回复突变株产胆固醇氧化酶的能力又明显下降,酶活分别为0.17 U/mL和0.69 U/mL.说明短杆茵 Brevibacterium sp.产胆固醇氧化酶能力与其产红色素成正相关偶联关系,这种相关性模型的建立可以作为以后诱变或定向进化研究的筛子.