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Significance. One of the modern trends in medical diagnostics is based on metabolomics, an approach allowing determination of metabolites which can be the specific features of disease. High-resolution gas spectroscopy allows investigation of the gas metabolite content of samples of biological origin. We present the elaboration of a method of studying diabetic and non-diabetic biological samples, prepared as pellets, by terahertz (THz) high-resolution spectroscopy. Aim: The main idea of the work is studying the content of thermal decomposition gas products of diabetic and non-diabetic dried blood plasma and kidney tissues for revealing the set of gas-markers that characterized the diabetes by the THz high-resolution spectroscopy method. Approach: We present an approach to study the diabetic and non-diabetic blood plasma (human and rats) and kidney tissues (rats), using high-resolution spectroscopy based on the non-stationary effect of THz frequency range. The methods of preparing the blood and kidney tissue samples as pellets and of vaporizing the samples were developed. Results: The measurements of rotational absorption spectra of vapors at heating the pellets prepared from blood and kidney tissue were carried out in 118 to 178 GHz frequency range. The absorption lines appearing in spectra of the sample vapors were detected and identified. The molecular contents of thermal decomposition products differed for non-diabetic and diabetic samples; e.g., main marker is acetone appearing in the diabetic blood (human and rats) and in the diabetic kidney tissue. Conclusions: Our paper illustrates the potential ability for determining the metabolite content of biological samples for diagnostics and prognosis of diseases for clinical medicine.

Significance: The creation of fundamentally new approaches to storing various biomaterial and estimation parameters, without irreversible loss of any biomaterial, is a pressing challenge in clinical practice. We present a technology for studying samples of diabetic and non-diabetic human blood plasma in the terahertz (THz) frequency range. Aim: The main idea of our study is to propose a method for diagnosis and storing the samples of diabetic and non-diabetic human blood plasma and to study these samples in the THz frequency range. Approach: Venous blood from patients with type 2 diabetes mellitus and conditionally healthy participants was collected. To limit the impact of water in the THz spectra, lyophilization of liquid samples and their pressing into a pellet were performed. These pellets were analyzed using THz time-domain spectroscopy. The differentiation between the THz spectral data was conducted using multivariate statistics to classify non-diabetic and diabetic groups’ spectra. Results: We present the density-normalized absorption and refractive index for diabetic and non-diabetic pellets in the range 0.2 to 1.4 THz. Over the entire THz frequency range, the normalized index of refraction of diabetes pellets exceeds this indicator of non-diabetic pellet on average by 9% to 12%. The non-diabetic and diabetic groups of the THz spectra are spatially separated in the principal component space. Conclusion: We illustrate the potential ability in clinical medicine to construct a predictive rule by supervised learning algorithms after collecting enough experimental data.

This study describes a microfluidic paper-based analytical device (μPAD) for separating plasma from whole blood and measuring glucose concentration. A two-dimensional μPAD was fabricated by wax printing, using chromatographic paper and was functionalized by the incorporation of chitosan into the plasma separation zone. Red blood cells were isolated from whole blood in the plasma separation zone consisting of a chitosan polymer structure and a wax barrier. The separated plasma, with buffer, was injected into the test zone to cause a color change. The colorimetric results were digitized with a scanner, and the concentration was calculated using a calibration curve. This μPAD, which separated the plasma from whole blood without a separation membrane, will be useful for constructing point-of-care testing devices that detect analytes in small sample volumes.

Plasma HIV viral load testing is the preferred means of monitoring antiretroviral treatment response. Dried blood spots (DBSs) hold considerable logistical advantages over EDTA samples, but they more frequently misclassify virological failure and have higher limits of detection (LoD)., Plasma HIV viral load testing is the preferred means of monitoring antiretroviral treatment response. Dried blood spots (DBSs) hold considerable logistical advantages over EDTA samples, but they more frequently misclassify virological failure and have higher limits of detection (LoD). Plasma separation cards (PSCs) may overcome these limitations. Health workers collected EDTA whole blood by venipuncture and 140 μl of finger-prick blood by capillary tube from 53 HIV-infected adults. Capillary blood was immediately transferred to PSCs. Additionally, 432 EDTA samples from HIV-infected adults were spotted onto PSCs and analyzed together with the finger-prick samples. Specificity and sensitivity of PSC with paired EDTA-PSC samples tested on a cobas 6800/8800 system with the cobas HIV-1 test (cobas HIV) was determined. LoD (3rd HIV-1 WHO International Standard) and stability at a range of temperatures and storage durations was determined using cobas HIV and cobas AmpliPrep/cobas TaqMan HIV-1 test v2.0 (CAP/CTM). Of 132 specimens with quantitative values for paired EDTA-PSC samples, the mean log10 difference between samples was 0.05 copies/ml (95% confidence interval [CI], −0.01 to 0.11). The LoD for cobas HIV was 790.2 copies/ml and for CAP/CTM was 737.9 copies/ml. At 1,000 copies/ml, PSC sensitivity was 97.0% (128/132) and specificity was 97.2% (343/353). Results correlated well with those from EDTA samples (Deming R2 = 0.90). PSC results were unaffected by temperature and storage conditions. PSC samples correlate well with plasma viral load and have adequate sensitivity and specificity. The improved performance may be as a result of a reduction in contribution from cell-associated viral nucleic acids. The card provides an alternative sample collection technology to DBSs.

Background: The association between oxidised low density lipoprotein (OxLDL) and cerebral infarction is suspected but not established. Objectives: To determine whether plasma OxLDL is a useful marker for monitoring oxidative stress in stroke patients. Methods: Plasma OxLDL concentrations were determined in 56 stroke patients with cerebral infarction (n = 45) or cerebral haemorrhage (n = 11), and in 19 age matched controls, using a novel sandwich enzyme linked immunosorbent assay. Results: Compared with the controls (0.130 (0.007) ng/µg LDL, mean (SEM)), OxLDL was significantly raised in patients with cerebral infarction (0.245 (0.022); p < 0.0001) but not in those with haemorrhage (0.179 (0.023)). Patients with cortical ischaemic infarcts (n = 22) had higher OxLDL levels than either the controls (p < 0.0001) or the patients with non-cortical ischaemic infarcts (n = 23) (p < 0.001). Increased OxLDL concentrations in patients with cortical infarcts persisted until the third day after stroke onset. The National Institutes of Health stroke scales in patients with cortical infarction were higher than in those with non-cortical infarction (p < 0.01). Conclusions: There is a significant association between raised plasma OxLDL and acute cerebral infarction, especially cortical infarction. Plasma OxLDL may reflect oxidative stress in stroke patients.

Blood samples from 34 workers at a pulp and paper mill and from 14 control persons were analysed for 2,3,7,8-substituted PCDDs and PCDFs. There were no statistically significant differences in total lipid-adjusted PCDD/PCDF concentrations, expressed as toxic equivalents, in blood plasma between the potentially exposed bleaching plant or paper mill workers and the controls. The mean level was 61 pg/g I-TEQ in bleaching plant workers, 60 pg/g I-TEQ in paper mill workers and 49 I-TEQ pg/g in controls. Regarding the concentrations of individual isomers, however, there was an indication that the blood plasma concentrations might be affected by the living and working environment.

This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase, ALP, and aspartate aminotransferase, AST) and total serum protein. A device consists of four components: (i) a top plastic sheet, (ii) a filter membrane, (iii) a patterned paper chip containing the reagents necessary for analysis, and (iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and “spiked” standards that are acceptable for routine clinical use. This device illustrates a type of test useable for a range of assays in resource-poor settings.

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.

We studied the use of dried spots of bodily fluids (plasma, whole blood, and mother's milk) on filter paper as a means of sample collection and storage for human immunodeficiency virus type 1 (HIV-1) viral load testing under stringent field conditions. Plasma placed directly in lysis buffer, which is customarily used for viral load assays, was used for comparison in all our experiments. Utilizing reconstruction experiments, we demonstrate no statistical differences between viral loads determined for plasma and mother's milk spotted on filter paper and those for the same fluids placed directly in lysis buffer. We found that the addition of whole blood directly to lysis buffer was unreliable and could not be considered a feasible option. However, viral load measurements for whole blood spotted onto filter paper correlated with plasma viral load values for both filter spots and lysis buffer (Pearson correlation coefficients, 0.7706 and 0.8155, respectively). In conclusion, dried spots of plasma, whole blood, or mother's milk provide a feasible means for the collection, storage, and shipment of samples for subsequent viral load measurement and monitoring. Virus material spotted and dried on filter paper is a good inexpensive alternative for collecting patient material to monitor the HIV-1 viral load. Measuring the HIV-1 burden from whole blood dried on filter paper provides a suitable alternative for low-technology settings with limited access to refrigeration, as can be found in sub-Saharan Africa.

Background Adiponectin is an adipocyte-derived hormone that acts as a marker of insulin sensitivity. Bloodspot sampling by fingerstick onto filter paper may increase the feasibility of large-scale studies of the determinants of insulin sensitivity. We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantifying adiponectin from dried blood spots and then demonstrate its application in a large trial (PROBIT). Methods We quantified adiponectin from 3-mm diameter discs (≈3 µL of blood) punched from dried blood spots obtained from: i) whole blood standards (validation); and ii) PROBIT trial samples (application) in which paediatricians collected blood spots from 13,879 children aged 11.5 years from 31 sites across Belarus. We examined the distribution of bloodspot adiponectin by demographic and anthropometric factors, fasting insulin and glucose. Results In the validation study, mean intra-assay coefficients of variation (n = 162) were 15%, 13% and 10% for ‘low’ (6.78 µg/ml), ‘medium’ (18.18 µg/ml) and 'high’ (33.13 µg/ml) internal quality control (IQC) samples, respectively; the respective inter-assay values (n = 40) were 23%, 21% and 14%. The correlation coefficient between 50 paired whole bloodspot versus plasma samples, collected simultaneously, was 0.87 (95% CI: 0.78 to 0.93). Recovery of known quantities of adiponectin (between 4.5 to 36 µg/ml) was 100.3–133%. Bloodspot adiponectin was stable for at least 30 months at −80°C. In PROBIT, we successfully quantified fasting adiponectin from dried blood spots in 13,329 of 13,879 (96%) children. Mean adiponectin (standard deviation) concentrations were 17.34 µg/ml (7.54) in boys and 18.41 µg/ml (7.92) in girls and were inversely associated with body mass index, fat mass, triceps and subscapular skin-fold thickness, waist circumference, height and fasting glucose. Conclusions Bloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood collected on filter paper and can be applied to large-scale studies.

This study evaluates the effects of handling and confinement stress and a 3-day recovery period on a number of biochemical parameters used to monitor exposure of fish to bleached kraft mill effluent (BKME). Plasma was collected at four times of the day from male and female white sucker subjected to four levels of handling stress during their spawning migration at a BKME and a reference site. Indicators of a general response to stress (plasma cortisol, glucose, lactate, and total protein) and of reproductive fitness (plasma testosterone, 11-ketotestosterone, and 17 beta-estradiol) were measured. With the exception of 17 beta-estradiol in females, all of the parameters measured varied with time of day and stress level. The general stress indicators ranged from being highly to marginally responsive to handling and confinement stress and were inconsistent in terms of indicating a site difference. The reproductive steroids were moderately responsive to stress and gave the most consistent site difference with testosterone and 11-ketotestosterone levels reduced in BKME-exposed fish under low levels of stress. There was limited evidence for recovery from capture, handling, and transport in the spring for plasma cortisol, lactate, and 17 beta-estradiol at the BKME site, plasma glucose at the reference site, and plasma testosterone at both sites. Fish were also more responsive to an additional acute stress on Day 1 and Day 3 of recovery compared to that on Day 0. This study emphasizes the need for standardized methods in field collections and sampling with the least amount of stress possible, and suggests that holding white sucker for 1 or 3 days does not allow them to recover from the stress of capture.

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24–55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15–22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.

Human urokinase (HUK) was purified from commercial product by high performance liquid chromatography on TSK GEL-G3000SW and affinity chromatography on benzamidine-Sepharose 4B. The purified enzyme was of a high molecular weight form (molecular weight of 53,000). This preparation was utilized as an antigen to immunize rabbits; the obtained antibody showed a high specificity against HUK. The antibody was conjugated to CNBr-activated paper disks. The antibody-conjugated paper disk and a fluorogenic peptide substrate, glutaryl-Gly-Arg-4-methyl-coumarine-7-amide, were used to measure urokinase (UK) activity in plasma. The calibration curve obtained by the proposed method passed through the origin and was linear in the range of 0-0.16 IU of HUK. The incubation of HUK with an excess amount of alpha 2-macroglobulin at 37 degrees C for 3 h gave only about a 30% decrease of the activity assayed by the proposed method. After incubations of HUK with alpha 1-antitrypsin and antithrombin III, the activity was completely inhibited. The incubation of HUK with plasma at 37 degrees C decreased the activity as a function of time. However, when the antibody-conjugated paper disk was used for the immunoreaction to HUK in plasma at 4 degrees C, no decrease of UK activity was observed. The plasma decay curve of UK activity after a single intravenous (i.v.) injection of HUK into a rabbit (12,000 IU/kg) indicated bi-exponential kinetics by using this assay method. The rate constants of the alpha and beta phases were 0.120 +/- 0.020 and 0.021 +/- 0.002 min-1, respectively. These result suggest that the proposed method is useful for measuring UK activity in plasma of patients with intravascular coagulation after i.v. administration of UK.

In a laboratory investigation fourhorn sculpin (Myoxocephalus quadricornis) were exposed for 5-9 months to wastewater from pine and birch lines from a bleached kraft pulp plant. This long-term exposure to bleached kraft mill effluent (BKME) affected the hematology, the ion balance, and the vertebral structure. Decreased values for hematocrit and hemoglobin and a reduced red blood cell count, as well as increased levels of methemoglobin, indicated disturbances in the red blood cell status. The white blood cell picture was not significantly affected by the BKME exposure. Decreased levels of potassium and chloride ions in the blood plasma in some of the exposed fish suggest an impaired ability to maintain ion homeostasis. Elevated frequencies of vertebral deformations in fish exposed to BKME confirm previous observations of vertebral damage in feral fourhorn sculpin caught in the receiving body of water of the same bleached kraft pulp industry. Many of the parameters analyzed in this investigation may be used as health indicators in future laboratory and field studies on fish exposed to BKME.

Rainbow trout ( Salmo gairdneri ) were exposed for 10 and 30 days to microbiologically treated effluent of a bleached kraft pulp and paper mill (BKME). Throughout the experiments, the quality of dilution water, taken from an upstream location close to the mill, was allowed to vary both diurnally and seasonally (September–October) in a natural manner. The test concentrations averaged 0 (control), 0.6, 1.2, 2.0, and 5.1% (v/v) of BKME. Conjugates of all major resin acids and chlorinated phenolics of BKME, except chlorocatechols, were detected in trout bile. They comprised 93.1–99.9% of the total amounts in the bile. The level of conjugated resin acids increased linearly with the content of BKME in test water. On the other hand, no increase in concentration of conjugated phenolics was seen above 2.0% BKME. In the blood plasma no conjugates were analyzable. Concentrations of free resin acids in the blood plasma, but not in the bile, significantly increased at high test concentrations of BKME. No active excretion of free resin acids from plasma to bile can be inferred, but some of the phenolics—particularly 3,4,5,6-tetrachloroguaiacol—seem to be concentrated in the bile as a free compound. Changes in several biochemical parameters did not display any simple relationship with the external concentrations of BKME. In intermediate exposure concentrations, however, liver RNA and protein concentrations were decreased after 10 days but increased after 30 days. Condition of fish, relative size of the liver, and concentrations of liver glycogen and water were unresponsive to BKME at the dilutions investigated.

1. 1. The protein composition of rat serum and whey was studied by paper electrophoresis in non-lactating rats and rats on the 13th and 20th days of lactation, and the turnover of serum proteins and their transfer to the milk were measured using 131I-labelled purified rat albumin, acidic α1-glycoprotein, haptoglobin, transferrin and γ-globulin. 2. 2. Lactation was accompanied by a decrease in serum albumin concentration and by increases of the α- and β-globulins. Whey contained lower concentrations of the electrophoretic fractions, but showed a marked increase in β-globulin between the 13th and 20th days of lactation. The fractional and absolute turnover rates of all five proteins were greater in the lactating than in the non-lactating rats, and all of the proteins were transferred to the milk. It is concluded that the rates of catabolism of all the proteins were increased during lactation due to loss into the milk, and that there was also an increase in their rates of synthesis. With the exception of transferrin the relative rates of transmission of the proteins to whey was in inverse relation to their molecular weight, suggesting that the mammary gland acts as a molecular sieve for serum proteins.

An ELISA for the quantitation of the C5a anaphylatoxin of complement in human plasma is presented which is based on the use of both a monoclonal antibody and polyclonal antibodies to C5a. Its detection limit is 1 ng C5a/ml plasma. It detects no C5a in fresh EDTA-plasma. Complete removal of C5 by an optimized precipitation step prior to the assay procedure is an essential feature of the method.

Summary The analysis of quinine in whole blood, plasma, urine, and samples dried on filter paper is described. Extraction was made with toluene followed by back-extraction into phosphate buffer. A reversed-phase liquid chromatography system with fluorescence detection was used. The within-day coefficient of variation of the method was 4–10% at the lower limit of determination (2 nM in plasma and 50 nM in whole blood, dried samples, and urine) and 2–4% at 10μM. The quinine concentration was found to be lower in whole blood than in plasma (mean ratio, plasma-whole blood, 1.17). The concentration in capillary blood was lower than that in venous blood (mean ratio, capillary blood-venous blood, 0.93).