Microliters Of Whole Blood Research Articles

Dried Blood Spots and Miniaturized Ultrasonic Nebulization Microplasma Optical Emission Spectrometry for Point-of-Care Testing of Blood Lithium.

Despite the significant importance of blood lithium (Li) detection in the treatment of bipolar disorder (BD), its point-of-care testing (POCT) remains a great challenge due to tedious sample preparation and the use of large-footprint atomic spectrometers. Herein, a system coupling dried blood spots (DBS) with a point discharge optical emission spectrometer equipped with a miniaturized ultrasonic nebulizer (MUN-μPD-OES) was developed for POCT of blood Li. Three microliters of whole blood were used to prepare a dried blood spot on a piece of filter paper to which 10 μL of eluent (1% (v/v) formic acid and 0.05% (v/v) Triton-X) was added. Subsequently, the paper was placed onto the vibrating steel membrane of the ultrasonic nebulizer and powered on to generate aerosol. The aerosol was directly introduced to the μPD-OES for quantification of Li by monitoring its atomic emission line at 670.8 nm. The proposed method minimized matrix interference caused by high levels of salts and protein. It is worth noting that the MUN suitably matches the needs of DBS sampling and can provide aerosolized introduction of Li into the assembled μPD-OES, thus eliminating all tedious sample preparation and the need for a commercial atomic spectrometer. Calibration response is linear in the therapeutic range and a limit of detection (LOD) of 1.3 μg L-1 is well below the Li minimum therapeutic concentration (2800 μg L-1). Li in mouse blood was successfully detected in real-time using MUN-μPD-OES after intraperitoneal injection of lithium carbonate, confirming that the system holds great potential for POCT of blood Li for patients with BD.

Estimation of reference interval for neutrophil activity evaluation systems: a interim report.

Neutrophils play an important role in innate immunity and produce reactive oxygen species, but they can also cause inflammation and oxidative stress that can damage their own tissues. We have developed neutrophil activity evaluation systems that simultaneously monitors superoxide radicals and hypochlorite ions secreted by stimulated neutrophils in a few microliters of whole blood and have conducted clinical studies in humans. Here, we report normal reference intervals with our systems based on the results of 3,082 persons who underwent comprehensive cancer screening between February 2020 and March 2022. A total of 344 were extracted as reference individuals based on the results of the cancer screening and the reference intervals of the two systems were interim estimated considering gender and age. Reference intervals can be used as a marker of sub-clinical inflam-mation, which is difficult to detect with other blood markers.

Comparison of the oxidative profiles before and after revascularization in peripheral arterial disease: a pilot study.

Reactive and highly reactive oxygen species (ROS and hROS) produced by white blood cells are essential for innate immunity; however, they may cause oxidative stress in the host. We developed systems for simultaneously monitoring ROS and hROS, i.e., superoxide radicals (O2•-) and hypochlorite ions (OCl-) secreted from stimulated white blood cells in a few microliters of whole blood. We previously reported on the evaluation of healthy volunteers' blood using the developed system; however, whether patients' blood can be assessed remains unclear. Here, we report a pilot study of 30 cases (28 patients) with peripheral arterial disease, in whom we measured the ROS and hROS levels before and approximately one month after endovascular treatment (EVT) using the system (CFL-H2200) that we developed. At approximately the same time points, physiological indices of blood vessels, oxidative stress markers, and standard clinical parameters in the blood were also monitored. The ankle-brachial index, a diagnostic tool for peripheral arterial disease, was significantly improved after EVT (p<0.001). The ROS-hROS ratio, low-density lipoprotein cholesterol, and hematocrit levels were decreased after EVT (p<0.05), while triglyceride and lymphocyte levels were increased after EVT (p<0.05). The correlations between the study parameters were also analyzed.

HAT-field: a cheap, robust and quantitative Point-of-care serological test for Covid-19.

The haemagglutination test (HAT)-field protocol described here is an optimization of the recently published HAT, for the detection of antibodies directed against the receptor binding domain (RBD) of the SARS-Cov-2 virus. HAT and HAT-field are both based on haemagglutination triggered by a single reagent, the IH4-RBD recombinant protein. A sample of IH4-RBD sufficient for several thousand tests or a plasmid encoding IH4-RBD can be obtained from the authors of our first paper. Using titration of IH4-RBD, HAT-field now allows a quantitative assessment of antibody levels in a single step, using a few microliters of whole blood, such as can be obtained by finger prick, and requires only very simple disposable equipment. Because it is based on a single soluble reagent, the test can be adapted very simply and rapidly to detect antibodies against variants of the SARS-CoV-2, or conceivably against different pathogens. HAT-field appears well suited to provide quantitative assessments of the serological protection of populations as well as individuals, and given its very low cost, the stability of the IH4-RBD reagent in the adapted buffer and the simplicity of the procedure, could be deployed pretty much anywhere, including in the poorest countries and the most remote corners of the globe.

Direct quantitation of tenofovir diphosphate in human blood with mass spectrometry for adherence monitoring.

Inadequate adherence to chronic medications is a far-reaching problem with financial and human health consequences. By a wide margin, non-adherence is the leading cause of therapeutic failures of HIV pre-exposure chemoprophylaxis (PrEP) and antiretroviral therapy (ART). It has been proven that HIV infection can be prevented by daily dosing of emtricitabine and tenofovir disoproxil fumarate. Measurement of intracellular tenofovir diphosphate in red blood cells has been established as an effective way to assess cumulative adherence, however, the LC-MS-based analytical method developed for the purpose is both complicated and expensive. Here, we report a simple method for adherence monitoring based on direct MS quantification of intracellular tenofovir diphosphate in human whole blood. The method requires only microliters of whole blood, employs special membranes to perform plasma separation and concomitant desalting during blood collection, and uses nanoelectrospray on a triple quadrupole instrument. Quantitative performance in this proof-of-concept study includes RSDs of < 15% and successful analysis of a small number of patient samples with medium to high adherence levels. The results correlate with those of a validated LC-MS/MS method, and an R2 value of 0.9962 is achieved. This methodology has promise for extension to point-of-care testing using miniature mass spectrometers. Graphical abstract.

Novel assay demonstrates that coronary artery disease patients have heightened procoagulant platelet response

Essentials Procoagulant platelets can be detected using GSAO in human whole blood.Stable coronary artery disease is associated with a heightened procoagulant platelet response.Agonist‐induced procoagulant platelet response is not inhibited by aspirin alone.Collagen plus thrombin induced procoagulant platelet response is partially resistant to clopidogrel. SummaryBackgroundProcoagulant platelets are a subset of highly activated platelets with a critical role in thrombin generation. Evaluation of their clinical utility in thrombotic disorders, such as coronary artery disease (CAD), has been thwarted by the lack of a sensitive and specific whole blood assay.ObjectivesWe developed a novel assay, utilizing the cell death marker, GSAO [(4‐(N‐(S‐glutathionylacetyl)amino)phenylarsonous acid], and the platelet activation marker, P‐selectin, to identify procoagulant platelets in whole blood by flow cytometry.Patients/MethodsUsing this assay, we characterized the procoagulant platelet population in healthy controls and a cohort of patients undergoing elective coronary angiography.ResultsIn patients with CAD, compared with patients without CAD, there was a heightened procoagulant platelet response to thrombin (25.2% vs. 12.2%), adenosine diphosphate (ADP) (7.8% vs. 2.7%) and thrombin plus collagen (27.2% vs. 18.3%). The heightened procoagulant platelet potential in CAD patients was not associated with other markers of platelet function, including aggregation, dense granule release and activation of α2bβ3 integrin. Although dual antiplatelet therapy (DAPT) was associated with partial suppression of procoagulant platelets, this inhibitory effect on a patient level could not be predicted by aggregation response to ADP and was not fully suppressed by clopidogrel.ConclusionsWe report for the first time that procoagulant platelets can be efficiently detected in a few microliters of whole blood using the cell death marker, GSAO, and the platelet activation marker, P‐selectin. A heightened procoagulant platelet response may provide insight into the thrombotic risk of CAD and help identify a novel target for antiplatelet therapies in CAD.

Sampling only ten microliters of whole blood for the quantification of poorly soluble drugs: Itraconazole as case study

Nowadays in animal studies, it is important to comply with the so-called Three Rs rule by replacing or reducing the number of tested animals. Volumetric absorptive microsampling (VAMS) can be used to collect small quantities (10 or 20μL) of whole blood, thereby limiting the amount of animals needed. In this study, a quantitative method was developed and subsequently validated for the poorly soluble drug itraconazole (ITZ) using VAMS and ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS). A proof of concept study showed that the optimized method is applicable to test the bioavailability of drug formulations containing ITZ. Using VAMS, smaller blood volumes can be taken per sampling point (10–20μL instead of the conventional 0.2-0.5mL) avoiding the sacrifice of animals. Moreover, the same rats can be used to compare different drug formulations which strengthens the validity of the results. In long-term bioavailability studies, it is necessary to guarantee the stability of the tested drugs supported on VAMS devices. In this study, we show that ITZ was only stable for 24h after collection with VAMS, but for at least two weeks by the storage of extracted samples at −80°C.

Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals.

Glutathione transferases (GSTs) are enzymes devoted to the protection of cells against many different toxins. In erythrocytes, the isoenzyme (e-GST) mainly present is GSTP1-1, which is overexpressed in humans in case of increased blood toxicity, as it occurs in nephrophatic patients or in healthy subjects living in polluted areas. The present study explores the possibility that e-GST may be used as an innovative and highly sensitive biomarker of blood toxicity also for other mammals. All distinct e-GSTs from humans, Bos taurus (cow), Sus scrofa (pig), Capra hircus (goat), Equus caballus (horse), Equus asinus (donkey) and Ovis aries (sheep), show very similar amino acid sequences, identical kinetics and stability properties. Reference values for e-GST in all these mammals reared in controlled farms span from 3.5±0.2 U/gHb in the pig to 17.0±0.9 U/gHb in goat; such activity levels can easily be determined with high precision using only a few microliters of whole blood and a simple spectrophotometric assay. Possibly disturbing factors have been examined to avoid artifact determinations. This study provides the basis for future screening studies to verify if animals have been exposed to toxicologic insults. Preliminary data on cows reared in polluted areas show increased expression of e-GST, which parallels the results found for humans.

Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species.

Neutrophil extracellular traps (NETs) were first reported in 2004, and since their discovery, there has been an increasing interest in NETs, how they are formed, their role in controlling infections, and their contribution to disease pathogenesis. Despite this rapid expansion of our understanding of NETs, many details remain unclear including the role of reactive oxygen species (ROS) in the formation of NETs. Further, to study NETs, investigators typically require a large number of cells purified via a lengthy purification regimen. Here, we report a microfluidic device used to quantify both ROS and NET production over time in response to various stimulants, including live bacteria. This device enables ROS and NET analysis using a process that purifies primary human neutrophils in less than 10 minutes and requires only a few microliters of whole blood. Using this device we demonstrate the ability to identify distinct capabilities of neutrophil subsets (including ROS production and NET formation), the ability to use different stimulants/inhibitors, and the ability to effectively use samples stored for up to 8 hours. This device permits the study of ROS and NETs in a user-friendly format and has potential for widespread applications in the study of human disease.

A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR

Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

Method development and validation for simultaneous quantification of 15 drugs of abuse and prescription drugs and 7 of their metabolites in whole blood relevant in the context of driving under the influence of drugs––Usefulness of multi-analyte calibration

In the context of driving under the influence of drugs (DUID), not only common drugs of abuse may have an influence, but also medications with similar mechanisms of action. Simultaneous quantification of a variety of drugs and medications relevant in this context allows faster and more effective analyses. Therefore, multi-analyte approaches have gained more and more popularity in recent years. Usually, calibration curves for such procedures contain a mixture of all analytes, which might lead to mutual interferences. In this study we investigated whether the use of such mixtures leads to reliable results for authentic samples containing only one or two analytes.Five hundred microliters of whole blood were extracted by routine solid-phase extraction (SPE, HCX). Analysis was performed on an ABSciex 3200 QTrap instrument with ESI+ in scheduled MRM mode. The method was fully validated according to international guidelines including selectivity, recovery, matrix effects, accuracy and precision, stabilities, and limit of quantification. The selected SPE provided recoveries >60% for all analytes except 6-monoacetylmorphine (MAM) with coefficients of variation (CV) below 15% or 20% for quality controls (QC) LOW and HIGH, respectively. Ion suppression >30% was found for benzoylecgonine, hydrocodone, hydromorphone, MDA, oxycodone, and oxymorphone at QC LOW, however CVs were always below 10% (n=6 different whole blood samples). Accuracy and precision criteria were fulfilled for all analytes except for MAM. Systematic investigation of accuracy determined for QC MED in a multi-analyte mixture compared to samples containing only single analytes revealed no relevant differences for any analyte, indicating that a multi-analyte calibration is suitable for the presented method. Comparison of approximately 60 samples to a former GC–MS method showed good correlation. The newly validated method was successfully applied to more than 1600 routine samples and 3 proficiency tests.

O112 How much does a non-physician healthcare worker based model of care for cardiovascular disease management cost?

Introduction: In patients with non-ST-elevation acute coronary syndrome (ACS), early risk stratification is of critical importance. Among others, the Thrombolysis in Myocardial Infarction (TIMI) risk score is the most validated and the most extensively used in patients with non-ST-elevation ACS. However, this score does not utilize results of evaluation of early markers of myocardial necrosis such as heart-type fatty acidbinding protein (hFABP). Objectives: The aim of this study was to estimate the effectiveness of new cardiac marker hFABP in the protocol of early risk stratification during rendering emergency medical care for patients with suspected non-ST-elevation acute coronary syndromes (NSTEACS). Methods: The study included two groups of patients. The first group included 442 patients (67 13 years) with suspected NSTEACS who underwent early risk stratification and the second (control group) consist of 491 patients (59 16 years) with suspected NSTEACS but without pre-operative risk stratification. We stratificated patient from first group accordind Modified TIMI risk score which was constructed with included results of hFABP evaluation. This evaluation was provided by a novel, sensitive assay that is based on the immunochromatographic test-strip technique has been recently developed for rapid qualitative detection of hFABP in whole blood (BioTest Co., Novosibirsk, Russia). The procedure requires approximately 100 microliters of whole blood and can be completed within 20 min in any in-patient or out-patient settings without any special equipment. The analytical sensitivity of this test is 15 ng/ml. Results: A preliminary diagnosis of myocardial infarction (MI) was adjudged in 52 (11.8%) patients from the first group and 64 (13.0%) from the second, and a preliminary diagnosis of unstable angina was adjudged in 390 (88.2%) and 427 (87.0%) patients, respectively. The accuracy of diagnosis of myocardial infarction in the first group was higher in the first group than the second (86,5% and 60,9% respectively, p1⁄40,003). The sensitivity of the hFABP assay within the first 12 hours was 84% for the detection of MI and the corresponding specificity was 93%. Conclusion: Using of new cardiac marker hFABP is effective in early risk stratification for patients with suspected non-ST-elevation acute coronary syndromes during rendering emergency medical care. Disclosure of Interest: None Declared

Morphological Features of Red Blood Cells in Patients with Severe Concomitant Injury

Objective: to reveal changes in the structure and shape of red blood cells depending on blood loss (BL) volume in patients with severe concomitant injury (SCI). Subjects and methods. Eighteen patients (9 men, 9 women) aged 48.6±16.1 years who had sustained severe concomitant mechanical injury (CMI) with different BL volumes and hemodynamic disorders were examined. According to the volume of BL, the patients were divided into two groups: 1) 7 victims with a BL volume of &lt; 750 ml (5.7±1.9 ml/kg), grade 1 BL; 2) 11 victims with a BL volume of &gt; 2000 ml (37.5±5.1 ml/kg), grade 4 BL. A comparison group consisted of 5 apparently healthy volunteers whose mean age was 26.4±2.7 years. The shapes and sizes of red blood cells were examined by light optical and atomic force microscopy (AFM). To study the composition of red blood cells, ten microliters of whole blood were applied to the slides and red blood cell monolayers were prepared using a V-sampler. The membrane surface was scanned by semicontact resonance AFM. The investigators used NSGL 01-A cantilevers with a resonance frequency in the range of 80200 kHz, a probe radius of 10 nm, 512 and 1024 scanning points, and 100X100 ^m and 10X10 ^m scanning fields. Planar and 3D images were obtained. Results. Calculation of 1000 cells by light optical microscopy and AFM showed significantly different counts of macro- and microcytes in the comparison group. The 100X100-^m field exhibited the following types of red blood cells: discocytes (97.9±1.5% and 96±5%), echinocytes (2.1±0.9% and 3±1%), and squamous cells (0.1±0.02% and 1±0.5%). Within the first 24 hours after injury, the victim group displayed lower normocyte counts and higher counts of macrocytes and microcytes than the control group. AFM in the 100X100-^m field revealed that the victims with SMI when admitted to an intensive care unit exhibited a significant decrease in the counts of discocytes counts and increases in those of echinocytes, stomatocytes, and squamous cells, as compared to the controls. Anisocytosis and poikylocytosis was found to depended on the degree of BL. The squamous cells are most likely to be a variety of young erythrocytes. An appreciable increase in macrocytes and squamous cells on day 5 most likely reflects enhanced compensatory erythropoiesis in response to BL. Examination of red blood cell shapes within the first 24 hours after injury in the AFM field revealed profound discocytes with a protuberance in the center. On day 1 of injury changes in the nanostructure of red blood cell membranes included: 9-fold increase of first-order height (hj); 1.5-fold and 3-fold increase of second- and third-order heights (h2 and h3), respectively. The magnitude of changes in hj, h2 and h3 depended on the volume of BL and tended to decrease within the first two weeks of injury. Conclusion. The victims with SMI were observed to possess significant anisocytosis and poikylocytosis. The changes were associated with the volume of BL and included profound discocytes with a protuberance in the center as well aso changes in heights of nanostructural patterns of red blood cell membranes. Key words: severe concomitant injury, blood loss, atomic force microscopy, light optical microscopy, nanostructure of red blood cell membranes.

Vitamin C in mouse and human red blood cells: An HPLC assay

Although vitamin C (ascorbate) is present in whole blood, measurements in red blood cells (RBCs) are problematic because of interference, instability, limited sensitivity, and sample volume requirements. We describe a new technique using HPLC with coulometric electrochemical detection for ascorbate measurement in RBCs of humans, wild-type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below the detection threshold (25nM). Twenty microliters of whole blood or 10μl of packed RBCs was sufficient for assay. RBC ascorbate was stable for 24h from whole-blood samples at 4°C. Processed, stored samples were stable for >1month at −80°C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R=0.8 and 0.9, respectively). In healthy humans, RBC ascorbate concentrations were 9–57μM, corresponding to ascorbate plasma concentrations of 15–90μM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately sevenfold and decreased 50% after 6weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in RBCs.

Whole Blood Assay for Elastase, Chymotrypsin, Matrix Metalloproteinase-2, and Matrix Metalloproteinase-9 Activity

The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 μL of 1× phosphate-buffered saline (PBS) (0.2-6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/mL were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases.

Integrated system investigating shear-mediated platelet interactions with von Willebrand factor using microliters of whole blood

We report an integrated platelet translocation analysis system that measures complex dynamic platelet–protein surface interactions in microliter volumes of unmodified anticoagulated whole blood under controlled fluid shear conditions. The integrated system combines customized platelet-tracking image analysis with a custom-designed microfluidic parallel plate flow chamber and defined von Willebrand factor surfaces to assess platelet trajectories. Using a position-based probability function that accounts for image noise and preference for downstream movement, outputs include instantaneous and mean platelet velocities, periods of motion and stasis, and bond dissociation kinetics. Whole blood flow data from healthy donors at an arterial shear rate (1500 s −1) show mean platelet velocities from 8.9 ± 1.0 to 12 ± 4 μm s −1. Platelets in blood treated with the antiplatelet agent c7E-Fab fragment spend more than twice as much time in motion as platelets from untreated control blood; the bond dissociation rate constant ( k off) increases 1.3-fold, whereas mean translocation velocities do not differ. Blood from healthy unmedicated donors was used to assess flow assay reproducibility, donor variability, and the effects of antiplatelet treatment. This integrated system enables reliable, rapid populational quantification of platelet translocation under pathophysiological vascular fluid shear using as little as 150 μl of blood.

Comparison of Results Obtained with Amplicor HIV-1 DNA PCR Test Version 1.5 Using 100 versus 500 Microliters of Whole Blood

The Amplicor HIV-1 DNA PCR assay (Roche Diagnostics, Branchburg, NJ) requires 500 microl of whole blood for a diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, and this amount is often difficult to obtain from infants. A comparison was performed using 100 and 500 microl of whole blood from infants less than 18 months of age. The concordance rate for HIV DNA PCR-negative and -positive samples was 100% for the two different volumes.

Blood in Saliva of HIV Seropositive Drug Abusers: Possible Implication in AIDS Transmission

We have studied hemoglobin concentration in saliva of anti-HIV positive and anti-HIV negative intravenous drug abusers (IVDA) and normal controls and the relationship between hemoglobin concentration in saliva and number of CD4+ cells and clinical status of AIDS in anti-HIV positive IVDA. 120 anti-HIV positive IVDA' 112 anti-HIV negative IVDA and 116 normal healthy subJects not belonging to any risk group for HIV infection completed the study. Saliva was collected at awakening before brushing teeth and the concentration of hemoglobin was determined. Hemoglobin concentration in saliva in basal conditions is higher in anti-HIV positive IVDA with respect to anti-HIV negative IVDA (p less than 0.05) and controls (p less than 0.01). In anti-HIV positive IVDA hemoglobin concentration in saliva is higher in subjects with CD4+ cells less than 200/10(6) l with respect to subjects with CD4+ greater than 200/10(6) l (p less than 0.05) and in subjects with ARC/AIDS with respect to subjects with PGL or who are asymptomatic (p less than 0.01). SubJects with ARC/AIDS have a mean concentration of hemoglobin of 19 micrograms/0.1 ml saliva (range 0–153) which corresponds to 1.3 microliters of blood/ml saliva. If 10 ml of saliva are exchanged during kissing an average of 13 microliters of blood are transferred (110 microliters of whole blood at extreme range). Blood of symptomatic patients has an HIV titer of 7 TCID/ microliters which for 10 ml saliva containing an average of 1.3 microliters blood/ml saliva corresponds to an average of 90 TCID (770 TCID at the extreme range). from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

A new handheld biosensor for monitoring blood ketones

A disposable amperometric biosensor for monitoring the blood ketones has been developed. It can be used to determine the concentration of β-hydroxybutyrate without pretreatment of blood samples. Enzymes are immobilized on screen-printed carbon electrodes. For immobilizing the enzymes, first a layer of hydrophilic gel CMC was printed on the screen-printed carbon electrodes. β-Hydroxybutyrate dehydrogenase, cofactor NAD and electron transfer mediator, potassium ferricyanide are immobilized on the electrode surface by adsorption. Experimentally the compositions of β-hydroxybutyrate dehydrogenase, potassium ferricyanide, NAD and other components are optimized. Amperometry performed by a handheld meter is used for the determination of β-hydroxybutyrate concentration. Two microliters of whole blood or serum is needed for one test, and the response time of the sensor is 50 s. Experimental results show that the responses of the sensors have relevant good linearity in the range 1.5–550 mg L −1. The testing results by the biosensors and the routine hospital blood analyzer are agreed closely.

Discovery stage pharmacokinetics using dried blood spots

Early in the discovery stage, the measurement of drug candidates in biological fluids as a function time provides important information used in decision making for lead optimization. The detection methodology primarily used is liquid chromatography coupled to triple quadrupole mass spectrometry (LC–MS). Sample preparation is an important aspect of these experiments and robotic-based automation is commonly used. The often overlooked aspect of these experiments is the sample collection itself. Typically, several hundred microliters of whole blood is collected and the plasma fraction separated for each time-point. The plasma is then transferred to an appropriate vessel for subsequent aliquoting and processing. We describe a method for performing discovery stage pharmacokinetic analysis using whole blood dried onto filter paper. The use of dried blood spots is a well established technique for neo-natal screening, and its application to early screening of drug candidates proves to be robust, reliable and reproducible.