Detection of progesterone in whole blood samples

The progesterone concentration in blood samples can be utilised as a marker for the diagnosis of early pregnancy, endocrinopathy and virilism. Here, we describe a method for progesterone detection and measurement in whole blood samples by a surface sensitive biosensor used in conjunction with an integrated optical grating coupler. This device determines refractive index changes near the biosensor's surface. Hence, biological species bound to a surface layer can be measured in real-time without any label. For the measurements, we have modified the indirect competitive immunoassay principle. The concentration of the progesterone antibody was kept at 1 microg/ml. Progesterone concentration was determined in buffer solution and whole blood in a range between 0.005 and 10 ng/ml. The detection limit was determined to be 3 pM. The relative standard deviation was calculated to be 3.5%.     

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.     

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.     

Headspace solid-phase microextraction in combination with gas chromatography and tandem mass spectrometry for the determination of organochlorine and organophosphorus pesticides in whole numan blood

A method for the determination of several organochlorine and organophosphorus pesticides in human whole blood samples was developed. The combination of solid-phase microextraction in headspace mode with gas chromatography with tandem mass spectrometry allowed the determination of 11 selected pesticides at ppb levels, minimizing the sample treatment. Quantitation was carried out by means of calibration curves prepared in blood using labelled surrogate/internal standards. The method showed good linearity between 1 and 50 ng ml(-1) (0.5-25 ng ml(-1) for HCB) using second-order calibration curves. Precision was found to be better than 20% at the three concentration levels assayed in the range of ng ml(-1). The detection limits obtained were in the range 0.02-0.7 ng ml(-1), except for p,p'-DDT (3 ng ml(-1)). The developed procedure was applied to blood and serum samples obtained from agricultural workers. HCB. beta-HCH and p,p'-DDE were most frequently detected in the samples analyzed.     

Comparative methodology for the detection and differentiation of circulating microfilariae of Dirofilaria immitis in the dog

The sensitivities of the Knott's test (four 20-microl sediment aliquots), quantitative buffy coat capillary tube method (QBC tube, 111 microl of whole blood) and direct blood smear (DBS, 20 microl of whole blood) were evaluated for the detection of microfilaraemia in dogs. Undiluted whole blood samples taken from 70 Dirofilaria immitis antigen-positive dogs and 10 serially diluted microfilaraemic blood samples at concentrations of 400, 200, 100, 50, 25 and 12 microfilariae (mff) ml(-1) were examined. For filarial speciation, the buffy coat of QBC tubes was mixed with one drop of methylene blue-formalin solution and examined as a direct smear. In 52/70 microfilaraemic blood samples, the number of mff ranged from 12 to 321987 ml(-1) (median: 3199 ml(-1)). The diagnostic sensitivity of the Knott's test, QBC tube method and DBS in undiluted blood samples attained the 100%, 98% and 92.3% levels, respectively. Eighteen dogs tested amicrofilaraemic by all three methods. At concentrations of 400 mff ml(-1), a 100% sensitivity was found by all three methods, while at 200 mff ml(-1) the Knott's test, QBC tube and DBS were 100%, 100% and 90% sensitive, respectively. The relevant figures at 100 mff ml(-1) were 100%, 100% and 80%, at 50 mff ml(-1) 100%, 100% and 50%, at 25 mff ml(-1) 100%, 100% and 10% and at 12 mff ml(-1) 80%, 50% and 10%. At 50 and 25 mff ml(-1), the DBS was less sensitive compared to the other two methods, while at 12 mff ml(-1), only to the Knott's test. A significant correlation was found between the QBC tube method and Knott's test regarding mff speciation. Therefore, the QBC method may be considered a reliable alternative to the Knott's test for both the detection and speciation of mff in the dog.     

Method for measuring hepatic uptake of oxygen or other blood-borne substances in situ.

A preparation is described by which hepatic arterial blood flow and portal venous blood flow can be accurately and continuously measured while simultaneously providing a method by which multiple blood samples can be taken from the hepatic artery, portal vein, and hepatic vein without disrupting hepatic hemodynamics or causing hemodilution. By this means hepatic uptake or release of blood-borne substances can be measured in situ and correlated with hemodynamic parameters. In 13 splenectomized cats, oxygen uptake by the denervated liver was 4.5 +/- 0.3 ml . min-1. 100 g-1 of tissue, representing 54% of total oxygen removed by the splanchnic bed. The hepatic hemodynamics determined by this method are similar to those reported by others in vivo and the metabolic state of the liver remained stable for at least 2 h during which an average of 29 blood samples were taken. Advantages of this preparation over other methods of obtaining similar data are discussed.     

Assessment of sirolimus concentrations in whole blood by high-performance liquid chromatography with ultraviolet detection

A novel, reversed-phase high-performance liquid chromatographic (HPLC) method is described for the analysis of sirolimus (SRL) in whole blood. The samples were purified by precipitating blood matrix with zinc sulfate, SRL was then extracted with acetone followed by solid-phase extraction. The method was linear over a range of 1-100 ng/ml and the lower limit of quantification was 2.5 ng/ml. The coefficient of variation (within day) was below 8.0% for the lowest SRL concentration. The day-to-day coefficient of variation was below 6.6%. The assay did not show interference peaks with immunosuppressive drugs commonly given to transplant patients. With the simplified extraction procedure described, 60 samples (including controls and calibration curve) can be quantified in a day. The sensitivity and rapidity of this analytical procedure makes it useful for routine therapeutic monitoring of SRL.     

A screening test to detect HGH-antibodies in pituitary dwarfs treated with HGH preparation by the radio-immunoassay with double antibody technique

Sixty-four blood samples, obtained from 25 pituitary dwarfs treated with KABIHGH preparation, were checked for serum HGH concentration by a double antibody radioimmunoassay and for antibodies against HGH by the method of propylethyleneglycol separation. Antibodies to HGH were detected in the samples whose HGH concentration was falsely estimated to be more than 5 ng/ml, but not in those whose HGH concentration was 5 ng/ml or less. The measurement of HGH concentration by a double antibody technique in the serum obtained from pituitary dwarfs 3-4 days after the last injection can be used to screen the presence of antibodies to HGH peparation used.     

Immunoenzyme determination of total serum ceruloplasmin. Application to Wilson disease

Enzyme immunoassay for ceruloplasmin (CP)*, employing monospecific CP antibodies labeled with horse radish peroxidase was developed. This method permits to determine total content of CP, which is present in Wilson disease patients' blood in enzymatically active and enzymatically inactive forms. The evidence is presented that the method can be used for a direct determination of CP in blood serum. The minimal CP concentration which may be determined by enzyme immunoassay (IEA) is 5.10(-9) g/ml. The method was used for determination of CP concentrations in Wilson disease patients' blood with different disease severity. Analysis of blood samples taken from 6 Wilson disease patients with the use of IEA method revealed similar total CP concentrations. At the same time, the oxidase activities of CP in the blood of different patients varied more than sevenfold.     

Techniques for collecting blood from collared peccaries, Dicotyles tajacu (L.)

Four methods are described for obtaining blood samples from the collared peccary. This animal lacks prominent superficial veins which makes the procedure of taking blood difficult for inexperienced persons. Large volumes of blood (greater than 20 ml) can be obtained easily via anterior vena cava venipuncture. Moderate amounts of blood (less than 20 ml) can be obtained from the orbital sinus. Lesser volumes of blood can be obtained from superficial veins located on the ear and the hind limb. The saphenous vein is distended easily due to its unique location across the cranial face of the tibia.     

Electrochemical determination of azidothymidine in human whole blood.

An electrochemical method based on differential pulse voltammetry is presented for the determination of AZT in whole blood of fasted subjects. A protein-free supernatant of whole blood is prepared using HClO4 precipitation followed by neutralization with phosphate buffer. The AZT is reduced at a hanging mercury drop electrode. The linear dynamic range of standards in buffer is from the detection limit of 4.1 nM to 206.5 microM (1.1 to 55,200 ng/ml). However, in spiked blood samples the linear dynamic range is from 0.029 to 0.29 microM (7.75 to 77.5 ng/ml). The whole blood assay yields a recovery of 92.30 +/- 5.92% compared to the standard solution assay. After a 30-min preparation time, each sample can be analyzed in 10 min by a manual procedure.     

Determination of corticosterone in mouse plasma by a sweeping technique using micellar electrokinetic chromatography

The separation and on-line concentration of corticosterone in mouse blood was achieved by means of capillary electrophoresis/UV absorbance using sodium dodecyl sulfate (SDS) as a surfactant. The procedure involved the use of an on-line sample concentration method by sweeping-micellar electrokinetic chromatography (sweeping-MEKC). Optimal on-line concentration and separation conditions were determined. The detection limit for this method was 5 ng/ml (S/N=3) and photodiode array detection at 247 nm was used for identification. For the analysis of actual samples, corticosterones from blood samples of a non-stressed and stressed mouse were determined. The results show that only a minor amount of corticosterone was produced by a non-stressed mouse, whereas a significant amount was present in the blood sample from a stressed mouse. The method developed here can be used to examine corticosterone levels as a marker of stress in test animals and may also be used for estimating the effect of stress-release medications.     

Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.     

Determination of dopexamine hydrochloride in human blood by high-performance liquid chromatography with electrochemical detection

A method is described for the determination of dopexamine hydrochloride at concentrations of 5 to 1000 ng/ml in human blood using electrochemical detection. The method uses a Hypersil ODS column and a mobile phase containing heptane sulphonate, orthophosphoric acid, diisopropylamine and disodium EDTA. Blood samples are stabilised immediately after collection by the use of dipotassium EDTA and a high concentration of sodium metabisulphite. The sample preparation procedure consists of a simple de-proteinisation with perchloric acid. The method is accurate, with inter-assay accuracies ranging from 100 to 104%, and is free of interference by blood from different individuals. Known and potential metabolites of dopexamine hydrochloride and a wide range of drugs do not interfere with the method. The method is precise with inter-assay coefficients on variation of 10.6% at 5 ng/ml and of less than 4.2% at higher concentrations. Stabilised blood samples may be stored for over six months at −25°C prior to analysis.     

Dried blood spot assay for estimation of metronidazole concentrations in rats and its application in single animal drug pharmacokinetic study

An HPLC-UV-dried blood spot (DBS) method for the estimation of metronidazole (MTZ) in rat whole blood is reported. Method employs Ahlstrom 226 sample collection paper and DBS samples were prepared by spotting with 30 μl of whole blood (spiked calibration standards/quality control samples/in vivo study samples). A 6mm disc was punched from each DBS and extraction was carried out using water containing the internal standard (tinidazole). The calibration for MTZ was linear over 2.5-50 μg/ml concentration range. Accuracy (% bias) and precision (expressed as % Coefficient of variation) values for within and between day were <20% at the lower level quality control sample (LQC) and <15% at all other concentrations tested. The limit of quantification (LOQ) of the method was 2.5 μg/ml. The validated method was applied for the analysis of in vivo pharmacokinetic (PK) study samples after intravenous administration of MTZ to a rat. Whole blood PK parameters observed in this study were in compliance with literature based PK parameters. The DBS sampling approach was found to be useful in a single animal pharmacokinetic study.     

Simple determination of cyclosporine in human whole blood by high-performance liquid chromatography

A simple and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of cyclosporine (CyA, also known as cyclosporin A) in human whole blood. The method entailed direct injection of the blood samples after deproteination using acetonitrile. Chromatography was carried out using an ODS column under isocratic elution with acetonitrile-5mM disodium hydrogen phosphate (75:25, v/v), pH 5.1 at 70 degrees C and a detector set at 210 nm. The mean absolute recovery of cyclosporine from blood was 97%, and the linearity was assessed in the range of 100-3000 ng/ml blood, with a correlation coefficient of greater than 0.999. The limit of quantification and detection of the present method were 100 and 50 ng/ml, respectively. This method has been used to analyze several hundred human blood samples for bioavailability studies.     

Electron ionization gas chromatography-mass spectrometric determination of residues of thirteen pyrethroid insecticides in whole blood

A new rapid and sensitive electron ionization gas chromatography-mass spectrometry method in selective ion monitoring mode (SIM) was developed for the determination of l3 synthetic pyrethroid insecticide molecules and their stereo isomers in whole blood. The pyrethroid insecticides investigated are allethrin, bifenthrin, cypermethrin, cyphonothrin, cyfluthrin, lambda-cyhalothrin, deltamethrin, fenvalerate, fenpropathrin, imiprothrin, permethrin, prallethrin and transfluthrin. The residues of pyrethroids are extracted from the whole blood using hexane and acetone mixture (80 + 20%) as solvent. All the pyrethroid residues were separated by using a gas chromatography-mass spectrometry operated in electron ionization mode and quantified in selective ion monitoring mode. The method can detect the residues of different pyrethroids down to the level 0.05-2 ng/ml. Recovery experiments conducted in whole blood samples at the fortification level 1-1000 ng/ml showed 91-103% recovery. The applications of the analytical method for the determination of pyrethroid residues in real samples were tested by analyzing 45 human blood samples collected from the population exposed continuously to different pyrethroid based formulations. The results are confirmed by spiking the known quantity of pyrethroids and subsequently their positive detection.     

Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h

The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease.     

Quantifying F2-isoprostanes in umbilical cord blood of newborn by gas chromatography-mass spectrometry

We attempted to improve the extraction procedures to determine the F(2)-isoprostanes in plasma of umbilical cord arterial and venous blood by gas chromatography mass spectrometry. Plasma samples were deproteinized and hydrolyzed; free and esterified F(2)-isoprostanes were extracted by solid-phase extraction columns with citric acid/methanol/cyclohexane and ammonia solution/methanol and then derivatized by PFBBr and BSTFA. Concentrations of total plasma F(2)-isoprostanes eluted at the retention time of an internal standard of 8-iso-prostaglandin F(2alpha)-D(4) were quantified. The absolute recovery was 83+/-1.9% (95% confidence). Intraassay precision and interassay precision were lower than 1.0%. Analytical accuracy was 99.0+/-0.4% (95% confidence). Linearity, r(2), over the concentration range of 10 to 5000 pg/ml of spiked 8-iso-prostaglandin F(2alpha) in plasma was 0.9985. The method detection limit was 21 pg/ml (99% confidence) and the limit of quantitation was approximately 4 pg/ml. Analysis of 200 neonatal cord blood samples revealed few overlapping peaks causing interference in the elution of the F(2)-isoprostanes. With the use of an autosampler and one technician, 48 samples can be completed within 24h with 6h of actual hands-on work. This method could be potentially employed for routine analysis of plasma F(2)-isoprostanes in clinical laboratories.     

Changes in the membrane permeability of erythrocytes in the presence of heparin

It has been found by the conductometric impulse method that membrane conductivity of human erythrocytes rises about 70 times at heparin concentration in blood samples above 0.2 mg/ml. The presence of edetic acid (EDTA) in blood samples inhibits the rise of membrane conductivity on addition of heparin.