Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

Dipeptidyl-amino-peptidase IV activity in stored buffy coat smears from human blood

The stability of dipeptidyl-amino-peptidase IV (DAP IV) activity in lymphoid cells of buffy coat smears from human blood was studied during storage for 40 days. Fixed or unfixed smears may be stored at 20 C for up to 24 hr before a decrease in activity occurs. Storage of either fixed or unfixed smears at 4 C, -10 C and -80 C results in a significant loss of activity within 24 hr. However, the cells retain more than 85% of their DAP IV activity for up to 10 days when stored fixed at -80 C. These data underscore the importance of proper processing of slides for DAP IV staining to avoid misinterpretation of results.     

Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation

Cord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells, generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition. DNA methylation PCs were associated with individual (P_PC1 = 1.4 × 10^?9; P_PC2 = 2.9 × 10^?5; P_PC3 = 3.8 × 10^-5; P_PC4 = 4.2 × 10^-6; P_PC5 = 9.9 × 10^-13, P_PC6 = 1.3 × 10^?11) and not with sample type (P_PC1-6>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired samples ranged from r = 0.66 to r = 0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (five sites had unadjusted P<10^?5). Estimated cell type proportions did not differ by sample type (P = 0.46), and estimated proportions were highly correlated between paired samples (r = 0.99). Differences in methylation and cell composition between buffy coat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.     

A rapid and quantitative coat protein complex II vesicle formation assay using luciferase reporters

The majority of protein export from the endoplasmic reticulum (ER) is facilitated by coat protein complex II (COPII). The COPII proteins deform the ER membrane into vesicles at the ER exit sites. During the vesicle formation step, the COPII proteins load cargo molecules into the vesicles. Formation of COPII vesicles has been reconstituted in vitro in yeast and in mammalian systems. These in vitro COPII vesicle formation assays involve incubation of microsomal membranes and purified COPII proteins with nucleotides. COPII vesicles are separated from the microsomes by differential centrifugation. Interestingly, the efficiency of the COPII vesicle formation with purified recombinant mammalian COPII proteins is lower than that with cytosol, suggesting that an additional cytosolic factor(s) is involved in this process. Indeed, other studies have also implicated additional factors. To facilitate biochemical identification of such regulators, a rapid and quantitative COPII vesicle formation assay is necessary because the current assay is lengthy. To expedite this assay, we generated luciferase reporter constructs. The reporter proteins were packaged into COPII vesicles and yielded quantifiable luminescent signals, resulting in a rapid and quantitative COPII vesicle formation assay.     

Erythrocyte concentrates, low in buffy coat: production, preservation and evaluation of its quality

Buffy coat-poor packed red cells were prepared from fresh ACD-, ACD-AG- and EDTA-blood, than resuspended with a preservation solution, containing glucose, adenine, guanosine, sucrose, citric acid and sodium citrate and stored at 4 degrees C for 6 weeks. The survival rate of resuspended red cells from ACD-AG-blood amounted to 77% after 6 weeks of storage. The ATP content of resuspended red cells was approximately 25% lower than in ACD-AG whole blood during storage caused probably by increased ATP consuming reactions at the red cell membrane. The P2G-content of resuspended red cells from ACD- and ACD-AG-blood decreased above 50% of the normal level during the first week, as fast as in ACD- and ACD-AG whole blood. The P2G-breakdown in red cells from EDTA-blood was delayed for a week due to the higher pH as in CPD blood. Additions of xylitol, inorganic phosphate, and bicarbonate in 6, 5 and 20 mM final concentrations in the red cell suspensions and an increased pH at the same time delayed the breakdown of ATP and P2G. Packed red cells can be administered fast enough at hematocrits to 0.60 that will be achieved by adding 50 to 100 ml preservation solution. Leukocytes and thrombocytes were reduceds to 70 to 80%. With increasing rate of reduction a higher loss of red cells occured. Buffy coat-poor red cell concentrate contains only few microaggregates. It diminishes the risc of febrile transfusion reactions and delays the appearance of alloimmunisation. The circulatory overload of patients is less frequent than after transfusions of red cell resuspensions containing a large resuspension volume.     

Molecular basis of HbE-beta-thalassemia and the origin of HbE in northeast Thailand: identification of one novel mutation using amplified DNA from buffy coat specimens

Amplification of DNA via polymerase chain reaction directly from a small amount of a buffy coat fraction was used to study the molecular basis of HbE-beta-thalassemia in the northeastern Thai population. Eight different mutations including the new one causing a beta o-thalassemia phenotype were detected. This novel mutation is an amber mutation at codon 26, which occurs at the same position as that of HbE; the most common hemoglobin variant in Southeast Asian countries. A pitfall in detection of the HbE mutation by restriction enzyme analysis was pointed out and differential diagnosis of the HbE mutation and the novel one by using allele specific oligonucleotide probes were described. Analysis of polymorphic restriction sites in the beta-globin gene cluster containing the beta E gene revealed two previously undescribed haplotypes in the Southeast Asian populations, which provide evidence for the multiple origins of beta E gene in Southeast Asian populations.     

Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.     

Evaluation of retroviral production systems using quantitative analysis

Because of the low titer of retroviral supernatant, it is necessary to develop and optimize large-scale retroviral production systems. To quantitatively determine the effect of a given operating condition (e.g., temperature and serum content) on producer cells' retrovirus-producing capacity, a mathematical model was used to analyze the static retroviral production system described by three processes: viral diffusion, decay, and generation. The analytical solutions of the defined model equations were fitted with experimental data to determine the specific retroviral production rate constant, which represents the competence of a retroviral production system. Two different retroviral production systems, inducible production of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped retrovirus from 293GPG/EGFP cells and constant production of ecotropic retrovirus from GP+E86/LNCX cells, were employed to demonstrate the feasibility of the engineering analysis. Our results indicated that the time-variant specific retroviral production rate of 293GPG/EGFP cells reached its maximum value of 5.7 x 10(-)(3) CFU/cm(2).h.cell, and the constant specific retroviral production rate of GP+E86/LNCX cells was 1.49 x 10(-)(2) CFU/cm(2).h.cell for the whole period of production. Furthermore, the effects of serum concentration and temperature on the ecotropic retroviral production system were examined separately. Our results suggest that producing ecotropic retroviruses with 10% fetal bovine serum at 37 degrees C is the optimal operating conditions for the long-term production system used here.     

Detection of quantitative trait Loci influencing recombination using recombinant inbred lines

The genetic basis of variation in recombination in higher plants is polygenic and poorly understood, despite its theoretical and practical importance. Here a method of detecting quantitative trait loci (QTL) influencing recombination in recombinant inbred lines (RILs) is proposed that relies upon the fact that genotype data within RILs carry the signature of past recombination. Behavior of the segregational genetic variance in numbers of chromosomal crossovers (recombination) over generations is described for self-, full-sib-, and half-sib-generated RILs with no dominance in true crossovers. This genetic variance, which as a fraction of the total phenotypic variance contributes to the statistical power of the method, was asymptotically greatest with half sibbing, less with sibbing, and least with selfing. The statistical power to detect a recombination QTL declined with diminishing QTL effect, genome target size, and marker density. For reasonably tight marker linkage power was greater with less intense inbreeding for later generations and vice versa for early generations. Generational optima for segregation variance and statistical power were found, whose onset and narrowness varied with marker density and mating design, being more pronounced for looser marker linkage. Application of this method to a maize RIL population derived from inbred lines Mo17 and B73 and developed by selfing suggested two putative QTL (LOD > 2.4) affecting certain chromosomes, and using a canonical transformation another putative QTL was detected. However, permutation tests failed to support their presence (experimentwise alpha = 0.05). Other populations with more statistical power and chosen specifically for recombination QTL segregation would be more effective.     

Detection of parent-of-origin effects for quantitative traits using general pedigree data

Genomic imprinting is a genetic phenomenon in which certain alleles are differentially expressed in a parent-of-origin-specific manner, and plays an important role in the study of complex traits. For a diallelic marker locus in human, the parental-asymmetry tests Q-PAT(c) with any constant c were developed to detect parent-of-origin effects for quantitative traits. However, these methods can only be applied to deal with nuclear families and thus are not suitable for extended pedigrees. In this study, by making no assumption about the distribution of the quantitative trait, we first propose the pedigree parental-asymmetry tests Q-PPAT(c) with any constant c for quantitative traits to test for parent-of-origin effects based on nuclear families with complete information from general pedigree data, in the presence of association between marker alleles under study and quantitative traits. When there are any genotypes missing in pedigrees, we utilize Monte Carlo (MC) sampling and estimation and develop the Q-MCPPAT(c) statistics to test for parent-of-origin effects. Various simulation studies are conducted to assess the performance of the proposed methods, for different sample sizes, genotype missing rates, degrees of imprinting effects and population models. Simulation results show that the proposed methods control the size well under the null hypothesis of no parent-of-origin effects and Q-PPAT(c) are robust to population stratification. In addition, the power comparison demonstrates that Q-PPAT(c) and Q-MCPPAT(c) for pedigree data are much more powerful than Q-PAT(c) only using two-generation nuclear families selected from extended pedigrees.     

Automated quantitative cytological analysis using portable microfluidic microscopy

In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide‐based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in‐suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample.

     

Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.     

High-throughput quantitative binding analysis of DNA aptamers using exonucleases

Aptamers are nucleic acid bioreceptors that have been used in various applications including medical diagnostics and as therapeutic agents. Identifying the most optimal aptamer for a particular application is very challenging. Here, we for the first time have developed a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity via the kinetics of aptamer digestion by exonucleases. We demonstrate the utility of this approach by isolating a set of new aptamers for fentanyl and its analogs, and then characterizing the binding properties of 655 aptamer–ligand pairs using our exonuclease digestion assay and validating the results with gold-standard methodologies. These data were used to select optimal aptamers for the development of new sensors that detect fentanyl and its analogs in different analytical contexts. Our approach dramatically accelerates the aptamer characterization process and streamlines sensor development, and if coupled with robotics, could enable high-throughput quantitative analysis of thousands of aptamer–ligand pairs.     

Direct fluorescent and indirect immunofluorescent assay in buffy coat for candidemia diagnosis in pediatric patients: a comparative study

The diagnosis of candidemia by blood culture has poor sensitivity; therefore, immunossupresed patients and those with risk factors may receive empirical antifungal therapy, wich is potentially toxic. Fluorescent tests have been developed to obtain an early and more sensitive diagnosis than blood culture. The aim of this study was to compare indirect immunofluorescence vs direct calcofluor white fluorescence in buffy coat for candidemia diagnosis. Sensitivity, specificity, predictive values, of positive and negative samples were 60%, 86%, 33%, 95% and 90%, 80%, 35%, 99%, for indirect immunofluorescence and calcofluor white, respectively.