Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA ""alleles"" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

Direct and Simultaneous Determination of Cd Cu, and Se in Blood Samples by Graphite Furnace Atomic Absorption Spectrometry

A graphite furnace atomic absorption spectrometric method is proposed for the direct and simultaneous determination of Cd, Cu, and Se in human blood. Samples were diluted 1:10 (v/v) in 0.5% (v/v) HNO(3) + 0.5% (v/v) Triton X-100 solution. For 12 mu L injected sample volume + 5 mu L, of 1000 mg L(-1) Pd(NO(3))(2) + 3 mu L of 1000 mg L(-1) Mg(NO(3))(2), the calculated characteristic masses (mo) were 0.9 pg Cd, 16 pg Cu, and 39 pg Se, which are close to those mo values for single-element conditions for THGA furnace (1.3 pg Cd, 17 pg Cu, and 45 pg Se). Calibration curves with linear correlations better than 0.999 were obtained. The limits of detection (LOD) were 0.03 mu g L(-1) Cd, 0.075 mu g L(-1) Cu and 0.3 mu g L(-1) Se, and the relative standard deviations (n= 12) were 2.5%, 0.3%, and 1.5%, respectively. The method was applied for Cd, Cu, and Se determination in 10 human blood samples and the results were in agreement at the 95% confidence level with those obtained by inductively coupled plasma mass spectrometry. Concentrations of analytes in the selected blood samples varied from 1.7 to 3.2 mu g L(-1) Cd, 700 to 921.7 mu g L(-1) Cu, and from 68.6 to 350 mu g L(-1) Se. The accuracy of the proposed method was also evaluated by an addition-recovery experiment and recoveries of Cd, Cu, and Se added to blood samples ranged from 99-109%, 91-103%,and 93-103%, respectively.

Western blotting method (TESAcruzi) as a supplemental test for confirming the presence of anti-Trypanosoma cruzi antibodies in finger prick blood samples from children aged 0-5 years in Brazil

Some Latin American countries have plans for total control and/or eradication of Chagas disease by the main vector (Triatoma infestans) and by blood transfusion. To achieve this, patients with Chagas disease must be identified. A Western blotting test, TESAcruzi, is described as a supplemental test for diagnosis of Chagas disease using samples collected from children <5 years living in different states of Brazil. Blood samples collected by finger prick on filter paper were sent to the test laboratory by a central laboratory to confirm results obtained previously. Ten percent of negative samples, all doubtful and all positive samples were received. Commercial reagents, IgG indirect immunofluorescence, enzyme immunoassay, and a recently introduced TESAcruzi test were used. From 8788 samples, 163 (1.85%) were reactive by IgG-ELISA and 312 (3.55%) by IgG IIF. From these, 77 (0.87%) were reactive in the TESAcruzi test. The results had high clinical value to identify those truly infected. (C) 2010 Elsevier B.V. All rights reserved.

Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae)

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

Non-Detection of Human Herpesvirus 8 (HHV-8) DNA in HHV-8-Seropositive Blood Donors from Three Brazilian Regions

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of all forms of Kaposi's sarcoma, primary effusion lymphoma and the plasmablastic cell variant of multicentric Castleman disease. In endemic areas of sub-Saharan Africa, blood transfusions have been associated with a substantial risk of HHV-8 transmission. By contrast, several studies among healthy blood donors from North America have failed to detect HHV-8 DNA in samples of seropositive individuals. In this study, using a real-time PCR assay, we investigated the presence of HHV-8 DNA in whole-blood samples of 803 HHV-8 blood donors from three Brazilian states (Sao Paulo, Amazon, Bahia) who tested positive for HHV-8 antibodies, in a previous multicenter study. HHV-8 DNA was not detected in any sample. Our findings do not support the introduction of routine HHV-8 screening among healthy blood donors in Brazil. (WC = 140).

Time course proteomic profiling of human myocardial infarction plasma samples: An approach to new biomarker discovery

Background: The aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology. Methods: Ten individuals with recent acute ischemic-type chest pain (< 12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T(0)) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T(2)), and the next four stages occurred at 12 h intervals after T(0). Individuals (n = 7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent). Results: Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p < 0.05). The 2STEMI group, had similar to 85% fewer differently expressed protein peaks than those without previous history of AMI (76, p < 0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T(0)), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM. Conclusions: Proteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery. (C) 2011 Elsevier B.V. All rights reserved.

Biomonitoring method for the simultaneous determination of cadmium and lead in whole blood by electrothermal atomic absorption spectrometry for assessment of environmental exposure

The aim of this work is to propose a biomonitoring method for the simultaneous determination of Cd and Pb in whole blood by simultaneous electrothermal atomic absorption spectrometry for assessment of environmental levels. A volume of 200 mu L of whole blood was diluted in 500 mu L of 0.2% (w v(-1)) Triton(R) X-100 + 2.0% (v v(-1)) HNO3. Trichloroacetic acid was added for protein precipitation and the supernatant analyzed. A mixture of 250 mu g W + 200 mu g Rh as permanent and 2.0% (w v(-1)) NH4H2PO4 as co-injected modifiers were used. Characteristic masses and limits of detections (n = 20, 3s) for Cd and Pb were 1.26 and 33 pg and 0.026 mu g L-1 and 0.65 mu g L-1, respectively. Repeatability ranged from 1.8 to 6.8% for Cd and 1.2 to 1.7% for Pb. The trueness of method was checked by the analysis of three Reference Materials: Lyphocheck(R) Whole Blood Metals Control level 1 and Seronorm(TM) Trace Elements in Whole Blood levels 1 and 2. The found concentrations presented no statistical differences at the 95% confidence level. Blood samples from 40 volunteers without occupational exposure were analyzed and the concentrations ranged from 0.13 to 0.71 mu g L-1 (0.32 +/- 0.19 mu g L-1) for Cd and 9.3 to 56.7 mu g L-1 (25.1 +/- 10.8 mu g L-1) for Pb. (C) 2007 Elsevier B.V. All rights reserved.

Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure

Despite the necessity to differentiate chemical species of mercury in clinical specimens, there area limited number of methods for this purpose. Then, this paper describes a simple method for the determination of methylmercury and inorganic mercury in blood by using liquid chromatography with inductively coupled mass spectrometry (LC-ICP-MS) and a fast sample preparation procedure. Prior to analysis, blood (250 mu L) is accurately weighed into 15-mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCI was added to the samples following sonication for 15 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 5 min on a C18 reverse-phase column with a mobile phase containing 0.05% (v/v) mercaptoethanol, 0.4% (m/v) L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% (v/v) methanol. The method detection limits were found to be 0.25 mu g L(-1) and 0.1 mu Lg L(-1) for inorganic mercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). The proposed method was also applied to the speciation of mercury in blood samples collected from fish-eating communities and from rats exposed to thimerosal. With the proposed method there is a considerable reduction of the time of sample preparation prior to speciation of Hg by LC-ICP-MS. Finally, after the application of the proposed method, we demonstrated an interesting in vivo ethylmercury conversion to inorganic mercury. (C) 2009 Elsevier B.V. All rights reserved.

Determination of total and inorganic mercury in whole blood by cold vapor inductively coupled plasma mass spectrometry (CV ICP-MS) with alkaline sample preparation

A simple method with a fast sample preparation procedure for total and inorganic mercury determinations in blood samples is proposed based on flow injection cold vapor inductively coupled plasma mass spectrometry (FI-CVICP-MS). Aliquots of whole blood (500 mL) are diluted 1 + 1 v/v with 10.0% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 3 h at room temperature and then further diluted 1 + 4 v/v with 2.0% v/v HCl. The inorganic Hg was released by online addition of L-cysteine and then reduced to elemental Hg by SnCl(2). On the other hand, total mercury was determined by on-line addition of KMnO(4) and then reduced to elemental Hg by NaBH(4). Samples were calibrated against matrix-matching. The method detection limit was found to be 0.80 mu g L(-1) and 0.08 mu g L(-1) for inorganic and total mercury, respectively. Sample throughput is 20 samples h(-1). The method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). For additional validation purposes, human whole blood samples were analyzed by the proposed method and by an established CV AAS method, with no statistical difference between the two techniques at 95% confidence level on applying the t-test.

Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure

A simple and fast method is described for simultaneous determination of methylmercury (MeHg), ethylmercury (Et-Hg) and inorganic mercury (Ino-Hg) in blood samples by using capillary gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) after derivatization and alkaline digestion. Closed-vessel microwave assisted digestion conditions with tetramethylammonium hydroxide (TMAH) have been optimized. Derivatization by using ethylation and propylation procedures have also been evaluated and compared. The absolute detection limits (using a 1 mu L injection) obtained by GC-ICP-MS with ethylation were 40 fg for MeHg and Ino-Hg, respectively, and with propylation were 50, 20 and 50 fg for MeHg, Et-Hg and Ino-Hg, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). Additional validation is provided based on the comparison of results obtained for mercury speciation in blood samples with the proposed procedure and with a previously reported LC-ICP-MS method. With the new proposed procedure no tedious clean-up steps are required and a considerable improvement of the time of analysis was achieved compared to other methods using GC separation.

Anemia screening in potential female blood donors: comparison of two different quantitative methods

Anemia screening before blood donation requires an accurate, quick, practical, and easy method with minimal discomfort for the donors. The aim of this study was to compare the accuracy of two quantitative methods of anemia screening: the HemoCue 201(+) (Aktiebolaget Leo Diagnostics) hemoglobin (Hb) and microhematocrit (micro-Hct) tests. Two blood samples of a single fingerstick were obtained from 969 unselected potential female donors to determine the Hb by HemoCue 201(+) and micro-Hct using HemataSTAT II (Separation Technology, Inc.), in alternating order. From each participant, a venous blood sample was drawn and run in an automatic hematology analyzer (ABX Pentra 60, ABX Diagnostics). Considering results of ABX Pentra 60 as true values, the sensitivity and specificity of HemoCue 201(+) and micro-Hct as screening methods were compared, using a venous Hb level of 12.0 g per dL as cutoff for anemia. The sensitivities of the HemoCue 201(+) and HemataSTAT II in detecting anemia were 56 percent (95% confidence interval [CI], 46.1%-65.5%) and 39.5 percent (95% CI, 30.2%-49.3%), respectively (p < 0.001). Analyzing only candidates with a venous Hb level lower than 11.0 g per dL, the deferral rate was 100 percent by HemoCue 201(+) and 77 percent by HemataSTAT II. The specificities of the methods were 93.5 and 93.2 percent, respectively. The HemoCue 201(+) showed greater discriminating power for detecting anemia in prospective blood donors than the micro-Hct method. Both presented equivalent deferral error rates of nonanemic potential donors. Compared to the micro-Hct, HemoCue 201(+) reduces the risk of anemic female donors giving blood, specially for those with lower Hb levels, without increasing the deferral of nonanemic potential donors.

Effectiveness of the CAGE questionnaire, gamma-glutamyltransferase and mean corpuscular volume of red blood cells as markers for alcohol-related problems in the workplace

Objective: To evaluate the usefulness of gamma-glutamyltransferase (GGT) and mean corpuscular volume (MCV), as well as that of the CAGE questionnaire, in workplace screening for alcohol abuse/dependence. Methods: A total of 183 male employees were submitted to structured interviews (Structured Clinical Interview for DSM-IV 2.0 and CAGE questionnaire). Blood samples were collected. Diagnostic accuracy and odds ratio were determined for the CAGE, GGT and MCV. Results: The CAGE questionnaire presented the best sensitivity for alcohol dependence (91%; specificity, 87.8%) and for alcohol abuse (87.5%, specificity, 80.9%), which increased when the questionnaire was used in combination with GGT (sensitivity, 100% and 87.5%, respectively; specificity, 68% and 61.5, respectively). CAGE positive results and/or alterations in GGT were less likely to occur among employees not presenting alcohol abuse/ dependence than among those presenting such abuse (OR for CAGE = 13, p < 0.05; OR for CAGE-GGT = 11, p < 0.05) or dependence (OR for CAGE = 76, p < 0.0 1; OR for GGT = 5, p < 0.0 1). Employees not presenting alcohol abuse/dependence were also several times more likely to present negative CAGE or GGT results. Conclusions: The use short, simple questionnaires, combined with that of low-cost biochemical markers, such as GGT, can serve as an initial screening for alcohol-related problems, especially for employees in hazardous occupations. The data provided can serve to corroborate clinical findings. (C) 2008 Elsevier Ltd. All rights reserved.

Blood and Salivary Oxidative Stress Biomarkers Following an Acute Session of Resistance Exercise in Humans

The aim of the present study was to compare oxidative stress biomarkers determined in blood and saliva before and after acute resistance exercise. 1 week after 1 maximum repetition (1RM) test 11 healthy well-trained males completed a hypertrophy acute session of resistance training including 3 sets of 10 repetitions at 75% of the 1RM, with 90s rest periods between sets. Venous blood and saliva samples were collected before (pre) and 10 min after (post) the resistance training session. A significant (p < 0.05) rise in blood lactate accumulation (pre: 1.6 +/- 0.4 vs. post: 9.5 +/- 2.4) was found post-acute resistance training compared with baseline values. Significant increases (p < 0.05) in TBARS (42%), AOPP (28%), uric acid (27%) and GSH (14%) were detected post-acute resistance training in relation to pre in blood samples. A significant increase (p < 0.05) in uric acid (36%) was found in saliva post-acute resistance training as well as a significant correlation (p < 0.05) between uric acid determined in blood and saliva. Statistical analysis did not reveal any other change in the salivary oxidative stress biomarkers. In conclusion, an acute session of resistance exercise induces oxidative stress in plasma of trained men after acute resistance training, which was not found in saliva samples except for uric acid.

Blood Cardioplegia with N-Acetylcysteine May Reduce Coronary Endothelial Activation and Myocardial Oxidative Stress

Objectives: The aim of this prospective study was to compare the efficacy of intermittent antegrade blood cardioplegia with or without n-acetylcysteine (NAC) in reducing myocardial oxidative stress and coronary endothelial activation. Methods: Twenty patients undergoing elective isolated coronary artery bypass graft surgery were randomly assigned to receive intermittent antegrade blood cardioplegia (32 degrees C-34 degrees C) with (NAC group) or without (control group) 300 mg of NAC. For these 2 groups we compared clinical outcome, hemodynamic evolution, systemic plasmatic levels of troponin I, and plasma concentrations of malondialdehyde (MDA) and soluble vascular adhesion molecule 1 (sVCAM-1) from coronary sinus blood samples. Results: Patient demographic characteristics and operative and postoperative data findings in both groups were similar. There was no hospital mortality. Comparing the plasma levels of MDA 10 min after the aortic cross-clamping and of sVCAM-1 30 min after the aortic cross-clamping period with the levels obtained before the aortic clamping period, we observed increases of both markers, but the increase was significant only in the control group (P=.039 and P=.064 for MDA; P=.004 and P=.064 for sVCAM- 1). In both groups there was a significant increase of the systemic serum levels of troponin I compared with the levels observed before cardiopulmonary bypass (P<.001), but the differences between the groups were not significant (P=.570). Conclusions: Our investigation showed that NAC as an additive to blood cardioplegia in patients undergoing on-pump coronary artery bypass graft surgery may reduce oxidative stress and the resultant coronary endothelial activation.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G I (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r = 0.62, P = 0.05), MAT(s) x MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r = 0. 14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. (c) 2007 Elsevier Ltd. All rights reserved.