Human Blood Specimens Research Articles

Determination of essential and toxic metals in blood by ICP-MS with calibration in synthetic matrix

A high throughput method has been developed and validated for the analysis of heavy metals in blood using inductively coupled plasma mass spectrometry (ICP-MS) with an integrated sample introduction system with discrete sampling (ISIS-DS). Blood analyses are performed using an Agilent 7700x ICP-MS in helium collision mode. For this study, we selected a metal panel that included As, Cd, Hg, Mn, Pb, and U. Blood specimens, reference material and intermediate calibration standards were diluted 50 times with a diluent composed of n-butanol, NH4OH, H4EDTA, Triton X-100 and five internal standards (ISTDs). A 50 times dilution alone however does not dissipate the blood matrix effect. We found that the addition of NaCl and CaCl2 to calibration standards created a synthetic matrix (SM) environment leading to excellent analytical accuracy for all metals in the study, with results for lead nearly identical to isotopic dilution technique results. In addition, our study demonstrated that under matrix-match conditions for blood samples and calibration standards, any ISTD can be selected for any analyte regardless of first ionization potential or atomic mass (i.e., 55Ge is an acceptable ISTD for 238U or 205Tl for 55Mn, etc.). This method was initially validated using the following external reference materials: four levels of NIST standard reference material (SRM) 955c samples supplied by the National Institute of Standards and Technology (NIST), Wisconsin State Laboratory of Hygiene (WSLH) blood sample pools that were used to prepare Filter Paper Blood Lead proficiency testing (PT) samples (June 2011 event), and multi-element blood samples supplied by the Institute National de Sante Publique Quebec (INSPQ). When calibration standards were prepared without the matrix-match components, notable differences between present study results and respective reference or mean values were observed. This approach may offer a universal technique to improve the accuracy of ICP-MS results for metal analyses in any complex matrix. This method is currently being used to analyze human blood specimens as part of the California Environmental Contaminant Biomonitoring Program (CECBP).

Utility and Limitations of Direct Multi-Locus Sequence Typing on qPCR-Positive Blood to Determine Infecting Leptospira Strain

Culture-independent molecular characterization of infecting Leptospira human blood specimens from a 2008 outbreak of human leptospirosis in central Sri Lanka was carried out. Of 58 quantitative real-time polymerase chain reaction-positive samples analyzed for seven multi-locus sequence typing (MLST) housekeeping genes (mreA, pfkB, pntA, sucA, tpiA, fadD, and glmU), interpretable data was obtained from 12 samples. Mean bacterial load was 2.2 × 10(5) among specimens with complete MLST profiles compared with 1.3 × 10(4) among specimens without complete MLST profiles; all specimens with complete profiles had at least 4.9 × 10(4) Leptospira/mL (t = 5, P < 0.001). Most (11/12) identified sequence types were ST1 (L. interrogans serovar Lai) and ST44 (L. interrogans serovar Geyaweera). MLST can be used to directly identify infecting Leptospira strains in blood samples obtained during acute illness without the need for culture isolation, but it shows important limitations related to bacterial load.

Analysis of lead toxicity in human cells.

BackgroundLead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity.ResultsWe analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high zinc protoporphyrin blood levels.ConclusionsThe results of our study defined specific changes in gene and protein expression in response to lead challenges and determined the injurious effects of exposures to lead on a cellular level. This information can be used for documenting the health effects of exposures to lead which will facilitate identifying and monitoring efficacious treatments for lead-related maladies.

Multispacer sequence typing relapsing fever Borreliae in Africa.

BackgroundIn Africa, relapsing fevers are neglected arthropod-borne infections caused by closely related Borrelia species. They cause mild to deadly undifferentiated fever particularly severe in pregnant women. Lack of a tool to genotype these Borrelia organisms limits knowledge regarding their reservoirs and their epidemiology.Methodology/Principal FindingsGenome sequence analysis of Borrelia crocidurae, Borrelia duttonii and Borrelia recurrentis yielded 5 intergenic spacers scattered between 10 chromosomal genes that were incorporated into a multispacer sequence typing (MST) approach. Sequencing these spacers directly from human blood specimens previously found to be infected by B. recurrentis (30 specimens), B. duttonii (17 specimens) and B. crocidurae (13 specimens) resolved these 60 strains and the 3 type strains into 13 species-specific spacer types in the presence of negative controls. B. crocidurae comprised of 8 spacer types, B. duttonii of 3 spacer types and B. recurrentis of 2 spacer types.Conclusions/SignificancePhylogenetic analyses of MST data suggested that B. duttonii, B. crocidurae and B. recurrentis are variants of a unique ancestral Borrelia species. MST proved to be a suitable approach for identifying and genotyping relapsing fever borreliae in Africa. It could be applied to both vectors and clinical specimens.

The pentosidine concentration in human blood specimens is affected by heating

Pentosidine is an advanced glycation end product, formed by oxidation and glycation that accumulates markedly during end-stage renal failure. Measurement of the pentosidine level in physiological samples is applied as a sensitive marker for the early diagnosis of renal failure. In the quantitative measurements of pentosidine reported to date, a rapid enzyme-linked immunosorbent assay (ELISA) has been widely used to estimate the plasma/serum pentosidine levels in a number of clinical samples, because high performance liquid chromatography (HPLC) methods require multiple preparation steps before the analysis. However, the currently used clinical analysis of the plasma/serum pentosidine level by ELISA requires incubation of the plasma/serum at 100°C for 15 min to inactivate the protease, which is required before the anti-pentosidine antibody can bind to the pentosidine. In the present study, we examined whether pentosidine could be generated artificially through the heating of serum. The pentosidine content, measured by HPLC, in the serum increased by heating in a temperature- and time-dependent manner. The pentosidine content was increased 1.1- to 4.2-fold by the heating process compared to unheated samples, and the increased rate was not identical for each sample. After removing low-molecular weight (<10,000) serum components, the heat-induced pentosidine formation was decreased. Furthermore, the increase in pentosidine formation was significantly inhibited by acidic conditions more than by the addition of diethylene triamine pentaacetic acid, a metal chelator. This indicates that the level of serum pentosidine will be measured more accurately by ELISA if hydrochloric acid is added during the heating process.

Biobanking human endometrial tissue and blood specimens: standard operating procedure and importance to reproductive biology research and diagnostic development

To develop a standard operating procedure (SOP) for collection, transport, storage of human endometrial tissue and blood samples, subject and specimen annotation, and establishing sample priorities. The SOP synthesizes sound scientific procedures, the literature on ischemia research, sample collection and gene expression profiling, good laboratory practices, and the authors' experience of workflow and sample quality. The National Institutes of Health, University of California, San Francisco, Human Endometrial Tissue and DNA Bank. Women undergoing endometrial biopsy or hysterectomy for nonmalignant indications. Collecting, processing, storing, distributing endometrial tissue and blood samples under approved institutional review board protocols and written informed consent from participating subjects. Standard operating procedure. The SOP addresses rigorous and consistent subject annotation, specimen processing and characterization, strict regulatory compliance, and a reference for researchers to track collection and storage times that may influence their research. The comprehensive and systematic approach to the procurement of human blood and endometrial tissue in this SOP ensures the high quality, reliability, and scientific usefulness of biospecimens made available to investigators by the National Institutes of Health, University of California, San Francisco, Human Endometrial Tissue and DNA Bank. The detail and perspective in this SOP also provides a blueprint for implementation of similar collection programs at other institutions.

Direct and Rapid Genotyping of Glutathione-S-transferase M1 and T1 from Human Blood Specimens Using the SmartAmp2 Method

Clinical studies have suggested that a defect in both glutathione S-transferase (GST) M1 and GSTT1 increases the risk of drug-induced hepatotoxicity. The present study developed the method that enables genotyping of GSTM1 and GSTT1 directly using a small aliquot of blood samples based on an isothermal Smart amplification process version 2 (SmartAmp-2). SmartAmp-2 reaction could complete the genotyping of GSTM1 and GSTT1 within 40 min. The frequency of wild-type, GSTM1 null, GSTT1 null, and double null was 24, 21, 35, and 19%, respectively, consistent with previous reports in the Japanese population. The genotypes of 94 human genomic DNA samples determined by SmartAmp-2 were identical to those determined by the conventional polymerase chain reaction method. SmartAmp-2 was able to determine the genotypes of GSTM1 and GSTT1 even when human blood specimens were used. The SmartAmp-2 method is a rapid and accurate means of identifying the GSTM1 and GSTT1 genotypes, making it less time and more labor efficient in clinical practice than conventional methods requiring preparation of genomic DNA and electrophoresis. This will contribute to evaluate the susceptibility of disease and adverse reactions to drugs caused by deletion of GSTM1 and GSTT1.

Antitumor and antimicrovascular effects of sorafenib (Soraf) in fresh human tumor culture in comparison with other putative tyrosine kinase inhibitors (TKI).

e13617 Background: Soraf is postulated to be a clinical anti-angiogenic agent (AAA). Yet preliminary clinical evidence suggests it may not be cross resistant with other AAAs, including sunitinib (Tamaskar, et al. J Urology 179:81,2008). An alternative hypothesis is that soraf has direct antitumor cell (ATC) effects. In order to determine whether antivascular (AV) or ATC effects were more important, we measured both AV and ATC effects of soraf in short term cultures of fresh human tumor biopsies and compared these activities with those of other putative TKIs. Methods: We used methods described in the following publication: Weisenthal, et al. Cell culture detection of microvascular cell death in clinical specimens of human neoplasms and peripheral blood. J Intern Med 264: 275–287, 2008. doi:10.1111/j.1365-2796.2008.01955.x Results: Utilizing methods described in Table 1 of the above publication, 2008, we determined the relative AV activity vs ATC activity of TKI and other agents. Expressed as a ratio of AV activity divided by ATC activity, the rank order (greatest to least relative AV activity) was as follows: pazopanib > bevacizumab > dasatinib > sunitinib > erlotinib > lapatinib > gefitinib > imatinib > sorafenib > cisplatin. AV activity of soraf was minimal. We also determined correlation coefficients (CC) for ATC activity of these agents (comparing activity of two agents at a time in the same specimen datasets; size of comparison TKI datasets ranging from 11 to 587, median of 247 direct activity comparisons between pairs of agents. CC are as follows: doxorubicin/epirubicin 0.88, cisplatin/carboplatin 0.86, paclitaxel/docetaxel 0.64, cisplatin/oxaliplatin 0.48, cisplatin/5FU 0.35, gefitinib/erlotinib 0.61, erlotinib/imatinib 0.51, erlotinib/lapatinib 0.46, erlotinib/dasatinib 0.41, erlotinib/sunitinib 0.28, sunitinib/imatinib 0.51, soraf/dasatinib 0.19, soraf/gefitinib 0.17, soraf/pazopanib 0.10, soraf/sunitinib 0.01, soraf/lapatinib 0.00, soraf/imatinib −0.01. Conclusions: Soraf has minimal AV activity in short term cultures of fresh human tumors, in comparison with other TKIs and has virtually non-cross resistant ATC activity. No significant financial relationships to disclose.

182. Regulation of PDX-1 Stability via Ubiquitination is Dependent Upon the Single Nucleotide Polymorphism (SNP) Status of Human Somatostatin Receptor Subtype 5 (SSTR5)

We have previously shown that SSTR5 is an important regulator of insulin expression/secretion and islet proliferation via pancreatic-duodenal homeobox 1(PDX-1) in mice. We have also identified a germline, non-synonymous single nucleotide polymorphism (SNP) of SSTR5 in human blood and pancreatic cancer specimens, which results in mutation of intracellular C-terminal proline 335 to leucine (SSTR5 P335L). The purpose of this study was to determine whether SSTR5 P335L SNP has any functional significance on PDX-1 expression and proliferation in human cell lines.

S20-04 Dose-related reference ranges for therapeutic drug monitoring in human blood specimens

A dose related reference range is calculated as a concentration range within that a drug concentration has to be expected in human blood specimens under medication with a given dose of the drug. The calculation is based on the direct correlation of the drug dose De (constant dose under long term therapy) to its blood concentration c with the total clearance of the drug (Clt) being the correlation coefficient (De = D / t = c × Clt). The clearance is taken as arithmetic mean ± standard deviation from phase II clinical trials of the drug. Thus the dose related reference range will statistically contain 68,27 % of all the drug concentrations determined under normal condition in the blood of a normal patient, where “normal” is defined as the patient population in the respective phase II clinical trial. It usually consists of patients 18-65 years of age without relevant comorbidity, comedication, and genetic abnormalities in drug metabolism. Therefore, any drug concentration determined outside its dose-related reference range creates a signal to alert for individual abnormalities in a patient such as drug-drug-interactions, gene polymorphisms that give rise to slow/rapid metabolizers, altered function of the excretion organs liver and kidneys by age and/or disease, compliance problems, a missing pharmacokinetic steady state and even signal overlay in the laboratory analysis. A table listing factors that allows the calculation of dose-related reference ranges for all relevant psychopharmaca will be included in the first update of the TDM consensus guideline to be published in Pharmacopsychiatry.

Dose-Related Reference Ranges for Therapeutic Drug Monitoring in Human Blood Specimens

A dose related reference range is calculated as a concentration range within that a drug concentration has to be expected in human blood specimens under medication with a given dose of the drug. The calculation is based on the direct correlation of the drug dose Dê (constant dose under long term therapy) to its blood concentration c with the total clearance of the drug (Clt) being the correlation coefficient (Dê=D/t=c x Clt). The clearance is taken as arithmetic mean±standard deviation from phase II clinical trials of the drug. Thus the dose related reference range will statistically contain 68,27% of all the drug concentrations determined under normal condition in the blood of a normal patient, where „normal“ is defined as the patient population in the respective phase II clinical trial. It usually consists of patients 18–65 years of age without relevant comorbidity, comedication, and genetic abnormalities in drug metabolism. Therefore, any drug concentration determined outside its dose-related reference range creates a signal to alert for individual abnormalities in a patient such as drug-drug-interactions, gene polymorphisms that give rise to slow/rapid metabolizers, altered function of the excretion organs liver and kidneys by age and/or disease, compliance problems, a missing pharmacokinetic steady state and even signal overlay in the laboratory analysis. A table listing factors that allows the calculation of dose-related reference ranges for all relevant psychoactive drugs will be included in the first update of the TDM consensus guideline to be presented by Hiemke et al. at the 8th workshop of the TDM working group of the AGNP (see Hiemke C et al in this issue).

Cell culture detection of microvascular cell death in clinical specimens of human neoplasms and peripheral blood

Angiogenesis studies are limited by the clinical relevance of laboratory model systems. We developed a new method for measuring dead microvascular (MV) cells in clinical tissue, fluid and blood specimens, and applied this system to make several potentially novel observations relating to cancer pharmacology. Dead MV cells tend to have a hyperchromatic, refractile quality, further enhanced during the process of staining with Fast Green and counterstaining with either haematoxylin-eosin or Wright-Giemsa. We used this system to quantify the relative degree of direct antitumour versus anti-MV effects of cisplatin, erlotinib, imatinib, sorafenib, sunitinib, gefitinib and bevacizumab. Bevacizumab had striking anti-MV effects and minimal antitumour effects; cisplatin had striking antitumour effects and minimal anti-MV effects. The ;nib' drugs had mixed antitumour and anti-MV effects. Anti-MV effects of erlotinib and gefitinib were equal to those of sunitinib and sorafenib. There was no detectable VEGF in culture medium without cells; tumour cells secreted copious VEGF, reduced to undetectable levels by bevacizumab, greatly reduced by cytotoxic levels of cisplatin + anguidine, and variably reduced by DMSO and/or ethanol. We observed anti-MV additivity between bevacizumab and other drugs on an individual patient basis. Peripheral blood specimens had numerous MV cells which were strikingly visualized for quantification with public domain image analysis software using bevacizumab essentially as an imaging reagent. This system could be adapted for simple, inexpensive and sensitive/specific detection of tissue and circulating MV cells in a variety of neoplastic and non-neoplastic conditions, and for drug development and individualized cancer treatment.

Rapid Detection of Hepatitis B Virus Surface Antigen by an Agglutination Assay Mediated by a Bispecific Diabody against Both Human Erythrocytes and Hepatitis B Virus Surface Antigen

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.

Simultaneous Analysis of Nitrite and Nitrate in Whole Blood by Ion Chromatography

A simple and modern method for simultaneous analysis of nitrite and nitrate in whole blood has been devised by ion chromatography with an autosuppressor and a conductivity detector. To our knowledge, this is the first report for such analysis dealing with whole blood as a specimen. A 1 mL volume of whole blood was mixed with 2 mL of Milli Q water, vortex‐mixed for hemolysis, and ultrafiltered to obtain a clear extract solution. It was passed through double connected mini-cartridges of silver resin and a silver absorbing material to remove a high concentration of chloride ion usually present in whole blood. An aliquot of the filtrate was analyzed on a Dionex DX‐500 instrument with a microbore IonPac® AS9‐HC column (2×250 mm). The present method gave very low backgrounds for blank whole blood at locations where nitrite should appear, and around the endogenous peak of nitrate. The spiked nitrite markedly decreased in whole blood, probably by the action of hemoglobin in vitro; it was only 10–20% 2 h after the addition. Therefore, quantitative validation had to be made only for nitrate. The recoveries of nitrate from whole blood were more than 90%; the linearity was confirmed in the range of 5–100 µg mL−1 for whole blood. Detection limit of nitrate in whole blood was about 0.5 µg mL−1. Coefficients of intra‐ and inter‐day variations were 3.66–7.14%. Using the present method, nitrite and nitrate concentrations were measured for human blood specimens after sniffing isobutyl nitrite gas in two male subjects. The endogenous nitrate concentrations without such sniffing were 1.27 to 7.60 µg mL−1 for seven subjects.

Prevalence and molecular analysis of Anaplasma platys in dogs in Lara, Venezuela

Blood specimens from clinically normal military dogs and their trainers, in Lara, Venezuela were screened for Anaplasma platys, A. phagocytophilum, or Ehrlichia ewingii using 16S rRNA PCR tests. Sixteen percent (7/43) of dog specimens were positive by A. platys PCR test followed by sequencing of the PCR products, and all human blood specimens [25] were negative. All specimens from these dogs and humans were PCR negative for E. ewingii or A. phagocytophilum. Twelve Rhipicephalus sanguineus ticks removed from these dogs were negative for A. platys by reverse transcription PCR test. Almost the entire 16S rRNA gene (1,364 bp) and groESL operon (1,646 bp) sequences of A. platys isolated from a dog were determined, revealing that both sequences were closely related to the sequences of an A. platys strain detected in R. sanguineus ticks from the Democratic Republic of Congo.

Peptidomic analysis of human blood specimens: Comparison between plasma specimens and serum by differential peptide display

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.

Simple extraction protocol for analysis of immunosuppressant drugs in whole blood.

The immunosuppressant drugs cyclosporine (cyclosporin A), tacrolimus, sirolimus, and everolimus are used to prevent organ rejection. Because commercial immunoassays are available only for cyclosporine and tacrolimus, HPLC (1)(2)(3), often in conjunction with mass spectrometry (MS or MS/MS) (4)(5)(6)(7), has been used to quantify these drugs. For MS/MS, sample preparation has often involved simple protein precipitation and solvent dissolution. Protocols have used zinc sulfate followed by acetonitrile (8), methanol (9), or acetone (10). Sometimes these reagents are premixed (11)(12)(13)(14). During in-house assay development we encountered several problems when zinc sulfate and acetonitrile were used. For example, many specimens clumped when blood was added to the zinc sulfate. Even after addition of acetonitrile and vortex-mixing, the clumps remained, necessitating manual dislodgement of the pellet. This occurred with lysed human blood specimens, lyophilized whole-blood controls (e.g., Bio-Rad Lyphochek), and College of American Pathologists proficiency testing specimens. We substituted methanol for acetonitrile with minor improvement. Another problem was that the recovery of sirolimus from whole blood using zinc sulfate followed by acetonitrile or methanol was <100% (9). In addition, the observed MS/MS responses for commercial calibrators and controls were often ∼30% higher than for human blood specimens. Here we present a new extraction protocol that improves absolute recoveries, provides excellent precision, and shows less ion suppression. Although the performance of this protocol for several immunosuppressants is described, we present example data only for sirolimus. We also describe a solid-phase extraction (SPE) that may be added to further enhance the cleanliness of extracts. For recovery and extraction experiments, we used cyclosporine from Novartis, tacrolimus from Fujisawa, and sirolimus from LC …

Three‐color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood

Experiments described herein were designed to evaluate the performance characteristics of a flow cytometry-based system that scores the incidence of peripheral blood micronucleated reticulocytes (MN-RETs). These procedures represent the continued refinement of a previously reported anti-CD71-based method (Dertinger et al. [1996]: Mutat Res 371:283-292), with the following modifications: incorporation of a third fluorescent label to exclude platelets from the MN-RET region, and use of a CD71-associated fluorescence thresholding technique to increase data acquisition rates. Mouse, rat, and human blood samples were analyzed using both the previously described two-color procedure (anti-CD71-FITC and propidium iodide) and a newly developed three-color technique (which adds an antiplatelet-PE antibody). The rodent specimens were also evaluated by standard microscopy procedures (acridine orange staining). Mouse blood was collected via heart puncture of vehicle- and 5-fluorouracil-treated CD-1 mice; blood samples from saline-treated Sprague-Dawley rats were collected from the tail vein and via heart puncture. Rodent blood samples were analyzed by both the two- and three-color methods. Human blood specimens, obtained via arm venipuncture from cancer patients undergoing radiation therapy, were analyzed for MN-RETs using the two-color method. Subsequently, blood samples from a single chemotherapy patient were analyzed by both the two- and three-color methods. Finally, the chemotherapy patient blood samples and blood samples from 15 healthy volunteers were evaluated at very high densities in conjunction with a CD71-associated fluorescence thresholding technique. Results of these investigations showed that data from mouse blood analyzed by the two- and three-color procedures correlated well with microscopy data (r values = 0.917 and 0.937 for the two- and three-color methods, respectively); all three methods confirmed the genotoxicity of 5-FU. Data from rat tail vein samples showed improved reproducibility with the three-color technique, but no significant difference between the two techniques was seen with the heart puncture specimens. Human blood analyzed according to the two-color procedure produced unreliable results, as platelets and platelet aggregates impacted the rare MN-RET scoring region. The three-color technique effectively overcame this problem and produced reproducible measurements that fell within expected ranges. For human blood analyses, the high cell density/CD71-thresholding technique provided significant improvements over the low-density technique, as it allowed data acquisition to occur approximately six times faster with no loss of sensitivity.

One-step immunoassay for acetaminophen and salicylate in serum, plasma, and whole blood.

Acetaminophen (APAP, n-acetyl-p-aminophenol) and salicylate (ASA, acetylsalicylic acid) are two of the most frequently overdosed drugs reported in the emergency room. Most APAP and ASA tests are performed with analytical instruments in laboratories, which are not always ideal for emergency situations. A rapid membrane and gold particle-based immunoassay for the simultaneous detection of APAP and ASA has been developed. Validation studies verified the cutoffs for the assay as 25 microg/mL for APAP and 100 microg/mL for ASA, respectively, and the accuracy study indicated that the agreement between the APAP and the ASA test and reference methods is 99%. One hundred thirty-three structurally unrelated chemicals and metabolites that may be present in human blood specimens were tested and found not to cross-react with the test.

An electrochemical detection scheme for identification of single nucleotide polymorphisms using hairpin-forming probes.

Single nucleotide polymorphisms are implicated as having a significant role in regulating growth, development and, thereby, human health and disease. We have developed a method for identifying single nucleotide genetic alterations by combining hairpin-forming DNA probes and electrochemical detection of sandwich DNA hybridization. Incorporation of hairpin-forming competitor probes and the catalyzed reporter deposition amplification system further improves assay specificity by 7-fold and sensitivity by 100-fold. We have demonstrated that the system successfully identified the factor V Leiden mutations from human blood specimens.