Human antibody immunosorption of hepatitis B surface antigen from blood and plasma

Removal of the Hepatitis B surface antigen (HBsAg) from whole blood and blood products, using human antibody (HBAb) immunosorbent, was studied and kinetics of complexing were monitored using radioimmunoassay (RIA). An intermittent complexing process was developed that minimizes damage to the cellular components of blood. HBsAg concentration in blood was reduced 1.5 to 2 logarithmic cycles in 3 hr with this system. Free HBsAg remaining in solution at equilibrium was further reduced by transferring the blood to a vessel containing unused immunosorbent. Through multiple stage treatment of a blood sample, it may be possible to reduce the probability of contamination with HBsAg to below the infectious level. This process may be applied to the selective removal of other proteins from blood and plasma.     

Renal extraction of glutamine from plasma and whole blood: studies in dogs and rats

The change in plasma and blood cell pools of L-glutamine during a single pass through the kidney was studied in dogs and rats. It was shown that the glutamine content of blood cells does not change following one passage through the renal vascular bed in normal or acidotic dogs. Furthermore, an infusion of L-glutamine elevating by 10-fold the plasma concentration of this amino acid only minimally changed the blood cells' glutamine content. Therefore within the time frame of acute experiments, the dog blood cells can be assumed to be impermeable to glutamine in vivo. Accordingly, renal glutamine extraction can be measured using either whole blood or plasma arteriovenous difference in this species. However, the latter value is larger and therefore can be measured more accurately. In normal rats, no net renal glutamine extraction is measured. In contrast, a considerable renal glutamine uptake occurs in acidotic rats, 23% of the extracted glutamine coming from the blood cell pool. A load of glutamine in vivo significantly elevates both the plasma and the blood cell concentration. It is concluded (i) that the renal extraction of glutamine is best estimated using plasma arteriovenous difference in the dog, especially when the renal extraction is small; (ii) that whole blood measurements should be obtained in the rat.     

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach.     

Comparing subjective and objective assessments of the severity of vibration induced white finger

The present staging of the disease severity of vibration induced white finger (VWF) is based on the patients' symptoms. Forty patients, with a history of VWF, with disease severity stage III or stage IV, on the Taylor-Pelmear scale, were investigated. Total, reactive hyperaemic blood flow to the hands was measured using an isotope limb blood flow (ILBF) technique. Skin blood flow patterns were assessed using a cold provocation test, followed by thermographic assessment of hand rewarming. Thermographic abnormalities were detected in 39 patients (97%). Decreased post-occlusive, reactive hyperaemic blood flow occurred in 29 patients (73%). There was no difference in skin blood flow patterns or in total hand blood flow between the stage III and stage IV groups. Reduction of postocclusive reactive hyperaemic blood flow may be indicative of occlusive lesions of the digital vessels. We conclude that the classification of the severity of VWF using subjective assessment, needs to be augmented by objective evidence of altered blood flow.     

A refined blood collection method for quantifying corticosterone

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.     

Fluorescence properties of rhodamine 800 in whole blood and plasma

We have characterized the fluorescence spectral properties of rhodamine 800 (Rh800) in plasma and blood in order to test the possibility of making clinical fluorescence measurements in whole blood without separation steps. Rh800 was used because of its absorption at red/near-infrared wavelengths away from the absorption bands of hemoglobin. We utilized the front-face illumination and detection to minimize the effects of absorption and/or scatter during measurements. The presence of Rh800 was detected in plasma and blood using steady-state fluorescence measurements. Absorption due to hemoglobin reduced the Rh800 intensity from the blood. Fluorescence lifetime measurements in plasma and blood showed that it is possible to recover lifetime parameters of Rh800 in these media. We obtained mean lifetimes of 1.90 and 1.86 ns for Rh800 in plasma and blood, respectively. Using the recently described modulation sensing method, we quantified the concentrations of Rh800 in plasma and blood. Rh800 was detected at a concentration of as low as 2 microM in both media. High anisotropy values were obtained for Rh800 in plasma and blood using steady-state and anisotropy decay measurements, implying the tight binding of this probe to the contents of these media. This binding can be exploited to monitor the concentrations of different blood components using already existing or new red-emitting probes that will be specially designed to bind to these components with high specificity. To test this possibility of direct measurements in blood, we used Rh800 to monitor albumin in the presence of red blood cells. Increase in the polarization of Rh800 as the concentration of albumin was increased in the presence of the red cells showed the feasibility of such measurements.     

Direct polymerase chain reaction (PCR) from human whole blood and filter-paper-dried blood by using a PCR buffer with a higher pH

We described a novel approach to directly amplify genomic DNA from whole blood and dried blood spotted on filter paper without any DNA isolation by using the PCR buffer with a higher pH, which was optimized as pH 9.1-9.6. Direct PCR on blood treated with various anticoagulants showed that the buffer worked well with the blood treated by citrate, EDTA, or heparinate. DNA fragments with different lengths could be efficiently amplified directly from various forms of blood samples. By coupling the buffer with tetra-PCR, a "true" single-tube genotyping was realized by using whole blood or paper-dried blood as starting material.     

Validation of Blood-Based Assays Using Dried Blood Spots for Use in Large Population Studies

Assessment of health in large population studies has increasingly incorporated measures of blood-based biomarkers based on the use of dried blood spots (DBS). The validity of DBS assessments made by labs used by large studies is addressed by comparing assay values from DBS collected using conditions similar to those used in the field with values from whole blood samples. The DBS approach generates values that are strongly related to whole blood levels of HbA1c, cystatin C, and C-reactive protein. Assessing lipid levels reliably with DBS appears to be a greater challenge. However, even when DBS values and values from venous blood are highly correlated, they are often on a different scale, and using conventional cutoffs may be misleading.     

Quantitative reconstruction of leukocyte subsets using DNA methylation

Background

Cell lineage-specific DNA methylation patterns distinguish normal human leukocyte subsets and can be used to detect and quantify these subsets in peripheral blood. We have developed an approach that uses DNA methylation to simultaneously quantify multiple leukocyte subsets, enabling investigation of immune modulations in virtually any blood sample including archived samples previously precluded from such analysis. Here we assess the performance characteristics and validity of this approach.

Results

Using Illumina Infinium HumanMethylation27 and VeraCode GoldenGate Methylation Assay microarrays, we measure DNA methylation in leukocyte subsets purified from human whole blood and identify cell lineage-specific DNA methylation signatures that distinguish human T cells, B cells, NK cells, monocytes, eosinophils, basophils and neutrophils. We employ a bioinformatics-based approach to quantify these cell types in complex mixtures, including whole blood, using DNA methylation at as few as 20 CpG loci. A reconstruction experiment confirms that the approach could accurately measure the composition of mixtures of human blood leukocyte subsets. Applying the DNA methylation-based approach to quantify the cellular components of human whole blood, we verify its accuracy by direct comparison to gold standard immune quantification methods that utilize physical, optical and proteomic characteristics of the cells. We also demonstrate that the approach is not affected by storage of blood samples, even under conditions prohibiting the use of gold standard methods.

Conclusions

Cell mixture distributions within peripheral blood can be assessed accurately and reliably using DNA methylation. Thus, precise immune cell differential estimates can be reconstructed using only DNA rather than whole cells.     

Mammary Blood Flow and Venous Drainage in Cows

The mammary blood flow and the udder drainage in vivo evaluated using the antipyrine absorption method has been compared with the anatomical findings in the udder after slaughtering of the experimental cows (Table 1). Because of the orientation of the valves in the perineal veins and blood samples taken in vivo it must be assumed that the perineal veins lead blood toward the veins at the udder base. It is concluded that the drainage of the udder in standing cows will primarily be through the milk veins, eventually there will be a flow of non-mammary venous blood down the external pudic veins at the udder base, as in the case of the perineal veins.     

Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Salmo gairdneri)

1. Total blood volume and relative blood volumes in selected tissues were determined in non-anesthetized, confined rainbow trout by using 51Cr-labelled trout erythrocytes as a vascular space marker. 2. Mean total blood volume was estimated to be 4.09 +/- 0.55 ml/100 g, or about 75% of that estimated with the commonly used plasma space marker Evans blue dye. 3. Relative tissue blood volumes were greatest in highly perfused tissues such as kidney, gills, brain and liver and least in mosaic muscle. 4. Estimates of tissue vascular spaces, made using radiolabelled erythrocytes, were only 25-50% of those based on plasma space markers. 5. The consistently smaller vascular volumes obtained with labelled erythrocytes could be explained by assuming that commonly used plasma space markers diffuse from the vascular compartment.     

Transport calculations for light scattering in blood.

In vivo measurement of the oxygen saturation levels in blood may be obtained from relative amounts of backscattered monochromatic light at two different wavelengths, as measured with a fiber-optic catheter oximeter. Because of the short mean free path length of light in blood, the backscattering can be well approximated by a previously-derived, one-wavelength transport theory solution for the half-space searchlight problem. This solution, unlike simple diffusion approximations has the advantage that the boundary condition describing illumination of a localized area of blood by a monodirectional light beam can be rigorously satisfied. Sample calculations using the solution are compared with experimental values of the reflectance of blood.     

Cost-effectiveness of blood agar for isolation of mycobacteria

Background

Mycobacterium species are grown using specific media that increase laboratory cost, thus hampering their diffusion in resource-limited countries. Preliminary data suggested that versatile blood agar may be also used for mycobacterial culture.

Methodology

We examined the growth of 41 different Mycobacterium species on 5% blood agar. Over a 24-month period we analysed isolation of mycobacteria after parallel inoculation of clinical specimens into both a reference automated system (BACTEC 9000 MB broth) and 5% blood agar slant tubes, after NaOH decontamination, and compared the cost of performing 1,000 analyses using these two techniques.

Conclusions

Mycobacterium reference species cultured on blood agar, with the exception of Mycobacterium ulcerans. Inoculation of 1,634 specimens yielded 95 Mycobacterium isolates. Blood agar performed significantly more efficiently than BACTEC 9000 MB broth (94 vs 88 isolates, P = 0.03). Decontamination of Candida albicans in 5 specimens by addition of amphotericin B in blood agar yielded one more M. tuberculosis isolate that could not be isolated in BACTEC broth. Uneven distribution of time to culture positivity for M. tuberculosis had a median (range) of 19±5 days using blood agar and 26±6 days using BACTEC 9000 MB broth. Cost for 1,000 analyses in France was estimated to be of 1,913 euros using the blood agar method and 8,990 euros using the BACTEC 9000 MB method. Blood agar should be regarded as a first-line medium for culturing Mycobacterium species. It saves time, is cost-effective, is more sensitive than, and at least as rapid as the automated method. This is of particular importance for resource-limited countries in which the prevalence of tuberculosis is high.     

Effects of prostaglandins on the affinity of hemoglobin for oxygen in human whole blood in vitro

The effect of prostaglandin on the affinity of hemoglobin for oxygen was tested on human blood $\text{in vitro}$, using six different prostaglandins at several dosage levels in fresh heparinized blood from normal donors and in stored citrated blood, and using prostaglandin E₂ on the blood from four seriously ill patients. No significant alterations in the affinity of hemoglobin for oxygen were dtected. A very small decrease in oxygen affinity in stored blood with high doses of prostaglandin was not statistically significant and would be of no physiologic significance even if real.We conclude that under the circumstances of this experiment prostaglandins do not alter the affinity of hemoglobin for oxygen in human whole blood $\text{in vitro}$.     

16. Environmental specimen cryobanking

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.     

Immunomagnetic bead separation of mononuclear cells from contaminating granulocytes in cryopreserved blood samples

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

A semi-empirical model for flow of blood and other particulate suspensions through narrow tubes

A semi-empirical model applicable to the flow of blood and other particulate suspensions through narrow tubes has been developed. It envisages a central core of blood surrounded by a wall layer of reduced hematocrit. With the help of this model the wall layer thickness and extent of plug flow may be calculated using pressure drop, flow rate and hematocrit reduction data. It has been found from the available data in the literature that for a given sample of blood the extent of plug flow increases with decreasing tube diameter. Also for a flow through a given tube it increases with hematocrit. The wall layer thickness is found to decrease with increase in blood hematocrit. A comparison between the results of rigid particulate suspensions and blood reveals that the thicker wall layer and smaller plug flow radius in the case of blood may be attributed to the deformability of the erythrocytes.     

Flurorometric study of the partition of bilirubin among blood components: basis for rapid microassays of bilirubin and bilirubin binding capacity in whole blood

Bilirubin binds to many sites in blood, the strongest binding being to a single site on albumin. Secondary sites on albumin, most sites on other plasma proteins, and sites on erythrocyte membranes have affinities for bilirubin that are at most one-hundredth as great. Bilirubin binds to hemoglobin in red cells with an effective affinity that is less than one-thousandth that of the primary albumin site. Essentially the only bilirubin present in blood which fluoresces is that bound to the primary albumin site. Almost all the other bilirubin in blood fluoresces with a yield no more than one-fiftieth as large. Quantitative fluorometry of whole blood is possible using the “front-face” technique. The concentration of bilirubin bound to the primary albumin site can be determined in this way. The albumin binding capacity of a blood specimen can be similarly assayed upon titration of the specimen with bilirubin. The nonionic detergent dodecyldimethylamine oxide (DDAO) scavenges bilirubin from all sites in blood, and, since bilirubin is fluorescent in DDAO micelles, the total blood bilirubin can be assayed fluorometrically after addition of DDAO to the specimen. This detergent method also allows facile assay of red-cell-bound bilirubin. These fluorometric assays for total blood bilirubin, albumin-bound bilirubin, and albumin binding capacity are simple and rapid and use very small volumes of blood. They should be of great value in the research on neonatal jaundice and in its clinical management.     

Investigation of volatile biomarkers in lung cancer blood using solid-phase microextraction and capillary gas chromatography-mass spectrometry

In the present work, solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was developed for investigation of lung cancer volatile biomarkers. Headspace SPME conditions (fiber coating, extraction temperature and extraction time) and desorption conditions were optimized and applied to determination of volatiles in human blood. To find the biomarkers of lung cancer, investigation of volatile compounds in lung cancer blood and control was performed by using the present method. Concentrations of hexanal and heptanal in lung cancer blood were found to be much higher than those in control blood. The two molecules of hexanal and heptanal were regarded as biomarkers of lung cancer. By comparison of volatiles in breath and in blood, it is demonstrated that hexanal and heptanal in breath were originated from blood and screening of lung cancer by breath analysis be feasible. These results show that SPME/GC-MS is a simple, rapid and sensitive method very suitable for investigation of volatile disease markers in human blood.