Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave an R2 of 0.95 for breath ammonia and BUN correlation and an R2 of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.

Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum.

Rare nucleated fetal cells circulate within maternal blood. Noninvasive prenatal diagnosis by isolation and genetic analysis of these cells is currently being undertaken. We sought to determine if genetic evidence existed for persistent circulation of fetal cells from prior pregnancies. Venous blood samples were obtained from 32 pregnant women and 8 nonpregnant women who had given birth to males 6 months to 27 years earlier. Mononuclear cells were sorted by flow cytometry using antibodies to CD antigens 3, 4, 5, 19, 23, 34, and 38. DNA within sorted cells, amplified by PCR for Y chromosome sequences, was considered predictive of a male fetus or evidence of persistent male fetal cells. In the 32 pregnancies, male DNA was detected in 13 of 19 women carrying a male fetus. In 4 of 13 pregnancies with female fetuses, male DNA was also detected. All of the 4 women had prior pregnancies; 2 of the 4 had prior males and the other 2 had terminations of pregnancy. In 6 of the 8 nonpregnant women, male DNA was detected in CD34+CD38+ cells, even in a woman who had her last son 27 years prior to blood sampling. Our data demonstrate the continued maternal circulation of fetal CD34+ or CD34+CD38+ cells from a prior pregnancy. The prolonged persistence of fetal progenitor cells may represent a human analogue of the microchimerism described in the mouse and may have significance in development of tolerance of the fetus. Pregnancy may thus establish a long-term, low-grade chimeric state in the human female.

A simple p53 functional assay for screening cell lines, blood, and tumors.

Mutations in the p53 gene are implicated in the pathogenesis of half of all human tumors. We have developed a simple functional assay for p53 mutation in which human p53 expressed in Saccharomyces cerevisiae activates transcription of the ADE2 gene. Consequently, yeast colonies containing wild-type p53 are white and colonies containing mutant p53 are red. Since this assay tests the critical biological function of p53, it can distinguish inactivating mutations from functionally silent mutations. By combining this approach with gap repair techniques in which unpurified p53 reverse transcription-PCR products are cloned by homologous recombination in vivo it is possible to screen large numbers of samples and multiple clones per sample for biologically important mutations. This means that mutations can be detected in tumor specimens contaminated with large amounts of normal tissue. In addition, the assay detects temperature-sensitive mutants, which give pink colonies. We show here that this form of p53 functional assay can be used rapidly to detect germline mutations in blood samples, somatic mutations in tumors, and mutations in cell lines.

Application of phosphatidylethanol (PEth) in whole blood in comparison to ethyl glucuronide in hair (hEtG) in driving aptitude assessment (DAA)

For driving aptitude assessment (DAA), the analysis of several alcohol biomarkers is essential for the detection of alcohol intake besides psycho-medical exploration. In Switzerland, EtG in hair (hEtG) is often the only direct marker for abstinence monitoring in DAA. Therefore, the suitability of phosphatidylethanol (PEth) was investigated as additional biomarker. PEth 16:0/18:1 and 16:0/18:2 were determined by online-SPE-LC-MS/MS in 136 blood samples of persons undergoing DAA and compared to hEtG, determined in hair segments taken at the same time. With a PEth 16:0/18:1 threshold of 210 ng/mL for excessive alcohol consumption, all (n = 30) but one tested person also had hEtG values ≥30 pg/mg. In 54 cases, results are not in contradiction to an abstinence as neither PEth (<20 ng/mL) nor hEtG (<7 pg/mg) was detected. In eight cases, both markers showed moderate consumption. Altogether, PEth and hEtG were in accordance in 68 % of the samples, although covering different time periods of alcohol consumption. With receiver operating characteristic analysis, PEth was evaluated to differentiate abstinence, moderate, and excessive alcohol consumption in accordance with hEtG limits. A PEth 16:0/18:1 threshold of 150 ng/mL resulted in the best sensitivity (70.6 %) and specificity (98.8 %) for excessive consumption. Values between 20 and 150 ng/mL passed for moderate consumption, values <20 ng/mL passed for abstinence. As PEth mostly has a shorter detection window (2-4 weeks) than hEtG (up to 6 months depending on hair length), changes in drinking behavior can be detected earlier by PEth than by hEtG analysis alone. Therefore, PEth helps to improve the diagnostic information and is a valuable additional alcohol marker for DAA.

Optimized methods for extracting circulating small RNAs from long-term stored equine samples.

Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Furthermore, we showed that miRNA of NGS-sufficient quality can be recovered from blood samples after >10 years of storage at -80 °C. This allows retrospective analyses of miRNAs from archived samples.

A method of sampling blood from Australian freshwater turtles

Blood sampling is an essential technique in many herpetological studies. This paper describes a quick and humane technique to collect blood samples from three species of Australian chelid turtles ( Order Pleurodira): Chelodina expansa, Elseya latisternum, and Emydura macquarii signata.

Misleading high tobramycin plasma concentrations can be caused by skin contamination of fingerprick blood following inhalation of nebulized tobramycin (TOBI (R)) - A short report

We observed unexpected high plasma concentrations of tobrarriycin (48.5 and 28.1 mg/L) in fingerprick blood samples after the nebulization of tobramycin solution for inhalation (tobramycin 300 mg/5 mL, TOBI(R)) by 2 young children aged 3 years. To investigate whether dermal contamination could be the source of error, 3 adult volunteers were present during another nebulization by a third child (age 2 years). The volunteers had exposure to tobramycin by handling the nebulizer or the nebule and also by inhalation from holding the child and being in close proximity while TOBI(R) was being administered. Five blood samples by fingerprick and 2 by venipuncture were collected and assayed for tobramycin concentration. On each occasion the site was swabbed with alcohol wipes to mimic standard patient sampling methods. One site was resampled after cleaning of hands with 2% chlorhexidine gluconate and water. Tobramycin concentrations from venipuncture 1-2 hours after nebulization were all < 0.2 mg/L except for 1 result of 1.2 mg/L. The tobramycin concentrations from fingerpricks before hand washing varied between 6.8 and 172 mg/L, and after hand washing between 0.3 and 17.6 mg/L. Contamination of fingers with tobramycin is likely to have caused the error in the 2 initial cases and did cause misleadingly elevated levels in the adult volunteers. We caution that therapeutic drug monitoring of nebulized tobramycin should not be done by fingerprick sampling, and care should be taken to avoid contamination of the venipuncture site.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 mu l) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3 -> 255.3). The assay was linear from 0.2 to 25 mu g/l (r(2) > 0.997, n = 5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 mu g/l) were 95.8-103.2 and < 5.5%, respectively. At the lower limit of quantification (0.2 mu g/l) the interand intra-day analytical recovery was 99.0-102.8% with imprecision of < 7.6% (n = 5). The assay had a mean relative recovery of 100.5 +/- 5.8% (n = 15). Extracted samples were stable for 16 h. IFTY720 quality control samples were stable at room temperature for 16 h at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans. (c) 2006 Elsevier B.V. All rights reserved.

Comparison of the reintroduced MEIA (R) assay with HPLC-MS/MS for the determination of whole-blood sirolimus from transplant recipients

Therapeutic monitoring with dosage individualization of sirolimus drug therapy is standard clinical practice for organ transplant recipients. For several years sirolimus monitoring has been restricted as a result of lack of an immunoassay. The recent reintroduction of the microparticle enzyme immunoassay (MEIA (R)) for sirolimus on the IMx (R) analyser has the potential to address this situation. This Study, using patient samples, has compared the MEIA (R) sirolimus method with an established HPLC-tandem mass spectrometry method (HPLC-MS/MS). An established HPLC-UV assay was used for independent cross-validation. For quality control materials (5, 11, 22 mu g/L), the MEIA (R) showed acceptable validation criteria based on intra-and inter-run precision (CV) and accuracy (bias) of < 8% and < 13%, respectively. The lower limit of quantitation was found to be approximately 3 mu g/L. The performance of the immunoassay was compared with HPLC-MS/MS using EDTA whole-blood samples obtained from various types of organ transplant recipients (n = 116). The resultant Deming regression line was: MEIA = 1.3 x HPLC-MS/MS+ 1.3 (r = 0.967, s(y/x) = 1) with a mean bias of 49.2% +/- 23.1 % (range, -2.4% to 128%; P < 0.001). The reason for the large and variable bias was not explored in this study, but the sirolimus-metabolite cross-reactivity with the MEIA (R) antibody could be a substantive contributing factor. Whereas the MEIA (R) sirolimus method may be an adjunct to sirolimus dosage individualization in transplant recipients, users must consider the implications of the substantial and variable bias when interpreting results. In selected patients where difficult clinical issues arise, reference to a specific chromatographic method may be required.

A novel dried blood spot-LCMS method for the quantification of methotrexate polyglutamates as a potential marker for methotrexate use in children

Objective: Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS). Methods: DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 μl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 μm, 2.1x150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization. Key Results: The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique. Conclusions and Clinical Relevance: The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology. © 2014 Hawwa et al.

An investigation of the relationship between antemortem and postmortem drug concentrations in blood

In the field of postmortem toxicology, principles from pharmacology and toxicology are combined in order to determine if exogenous substances contributed to ones death. In order to make this determination postmortem and (whenever available) antemortem blood samples may be analyzed. This project focused on evaluating the relationship between postmortem and antemortem blood drug levels, in order to better define an interpretive framework for postmortem toxicology. To do this, it was imperative to evaluate the differences in antemortem and postmortem drug concentrations, determine the role microbial activity and evaluate drug stability. Microbial studies determined that the bacteria Escherichia coli and Pseudomonas aeruginosa could use the carbon structures of drugs as a source of food. This would suggest prior to sample collection, microbial activity could potentially affect drug levels. This process however would stop before toxicologic evaluation, as at autopsy blood samples are stored in tubes containing the antimicrobial agent sodium fluoride. Analysis of preserved blood determined that under the current storage conditions sodium fluoride effectively inhibited microbial growth. Nonetheless, in many instances inconsistent drug concentrations were identified. When comparing antemortem to postmortem results, diphenhydramine, morphine, codeine and methadone, all showed significantly increased postmortem drug levels. In many instances, increased postmortem concentrations correlated with extended postmortem intervals. Other drugs, such as alprazolam, were likely to have concentration discrepancies when short antemortem to death intervals were coupled with extended postmortem intervals. While still others, such as midazolam followed the expected pattern of metabolism and elimination, which often resulted in decreased postmortem concentrations. The importance of drug stability was displayed when reviewing the clonazepam/ 7-aminoclonazepam data, as the parent drug commonly converted to its metabolite even when stored in the presence of a preservative. In instances of decreasing postmortem drug concentrations the effect of refrigerated storage could not be ruled out. A stability experiment, which contained codeine, produced data that indicated concentrations could continue to decline under the current storage conditions. The cumulative data gathered for this experiment was used to identify concentration trends, which subsequently aided in the development of interpretive considerations for the specific analytes examined in the study.

Haematological and biochemical blood study in baby alpaca with diarrhoea

The aim of this study was to determine the haematological value and biochemical blood in baby alpacas with enteric disorder. A total of 30 blood and serum samples were collected from alpacas of 1 month old with diarrhoea and 5 blood samples of clinically healthy baby alpacas (controls). The animals were from communities in the central Andes from Peru. About haematology were determined haematocrit, haemoglobin concentration, red blood count and white blood count that were not significantly different between control animals and animals with diarrhoea. Moreover, biochemical blood parameters as total protein, albumin and calcium decrease significantly (p<0.05). We conclude that our results could be considered as factors in the mortality of baby alpaca by infectious diarrhoea.

A comparison of different pre-lysis methods and extraction kits for recovery of Stretococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood

Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidasepre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6–85.1, p < 0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2–8.9, p < 0.001), compared with no pre-lysis. Differences in yield dueto pre-lysis were 2–3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture.

A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples

Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.

A study on cytochemical properties of white blood cells from Acipenseridae of southern Caspian Sea

The objective of this study was to study leukocytes characteristics of Acipenseridae of southern Caspian Sea stained by cytochemical stains in order to classify these cells more accurately. The samples were taken from Sturgeon propagation centers and International Sturgeon Research Institute in the north part of Iran. 10 fish were selected randomly from each age group (fingerling, 1, 2, 3 to 11 years) and blood samples were taken from caudal vein using syringes with no anticoagulant. Blood smears were prepared immediately and fixed by methanol. The smears then stained using cytochemical staining according to the Sigma-Aldrich instructions. Neutrophils were positive for Sudan Black-B(SBB), Periodic acid-Schiff (PAS) and Peroxidase (PER) but negative for choloroacetate esterase (CAE). Eosinophi is stained positively for PER and SBB and weakly for PAS, acid phosphatase (ACP), CAE and a-naphthyl acetate esterase (a-NAE). Basophil was absent in the studied blood smears. Lymphocytes were positive for ACP but weak for PAS, CAE and a-NAE. Monocytes stained positively for B-glucoronidase (BG) in Acipenser persicus 5 and 11 years group, in Acipenser stellatus 1 and 2 years group, in Acipenser gueldenstaedtii 1 year group, in Acipenser nudiventris 9 year and in Huso huso 7 year group and stained weakly for a-NAE.