Effect Of Hematocrit Research Articles

Development of an enzyme-linked immunosorbent assay for newborns dried blood spot thyroglobulin.

Thyroglobulin (Tg) is a biomarker of iodine status. Newborn Tg is a more sensitive marker than neonatal TSH in detecting variations in iodine intake. This study aims to validate a Tg enzyme-linked immunosorbent assay (ELISA) for Tg determination on dried blood spots (DBS) in newborns. This study also set out to assess the stability of Tg and the influence of newborns' hematocrit on Tg determination. A commercially available ELISA Tg assay was adapted for use on DBS. DBS-Tg in cord blood were measured in 209 newborns delivered from healthy euthyroid pregnant women. Sensitivity, linearity, repeatability, and intermediate fidelity were determined using the appropriate standards and quality control materials. The limit of detection (LoD) of the DBS-Tg assay was 2.4 µg/L, and the limit of quantification (LoQ) was 5.8 µg/L. Repeatability and intermediate fidelity were 7.7-8.3% and 11.0-11.2%, respectively. The median cord plasma Tg and DBS-Tg values in newborns were not significantly different, 30.2 (21.3-44.4) µg/L and 31.6 (19.3-48.7) µg/L (p=0.48) with the ELISA respectively, and 76.5 (40.0-101.5) µg/L with the Elecsys assay with an R=0.88. DBS-Tg concentrations decrease with increasing hematocrit values (p<0.05). DBS-Tg values were stable at a concentration of 25 µg/L for 12 months at -20ºC and 4ºC. This DBS-Tg assay demonstrated good analytical performances over a wide range of Tg concentrations, suggesting it is well suited to detecting variations in Tg concentrations. Studies comparing populations with different prevalence of anemia should consider the effect of hematocrit on DBS-Tg determination. The availability of a DBS-Tg assay for newborns makes it possible to integrate iodine status monitoring with newborn screening for inherited metabolic diseases.

On the Use of Image Analysis for Hematocrit Evaluation in Dried Blood Spots

Dried blood spots (DBSs) are formed by collecting a small sample of blood on specialized filter paper and allowing it to dry naturally. Various domains of life sciences and drug research extensively use DBSs as a sampling technique. The “Hematocrit (Ht) effect” affects assay bias, and several strategies have been put forth to deal with it, including the correction of quantified concentrations using an appropriate correction factor. The approach was previously applied, following the utilization of an image processing algorithm developed in Matlab® to derive a reliable equation correlating DBS areas to Ht% values. The present work looks more closely at the application of image analysis to the evaluation of Ht in DBS samples. Utilizing image analysis software, DBS samples with known Ht values were processed. Preparation of cards has followed a previously developed protocol for the appropriate formation of uniform area DBSs, irrespective of Ht. The resulting areas showed close resemblance to the respective theoretical areas calculated by applying the correlation equation. Following that, the equation was utilized to determine the Ht values for each sample, and a comprehensive comparison of measured versus calculated Ht was carried out using various statistical approaches for method comparison. The results demonstrated a strong correlation, suggesting the method’s viability in estimating Ht for unknown DBS samples.

Management of anticoagulant-related soft tissue bleeding

Spontaneous, severe, and life-threatening soft tissue bleeding (STB) in patients taking anticoagulants is associated with high morbidity and mortality due to the substantial blood loss and nonspecific clinical symptoms. The optimal management of these predominantly older patients with multiple comorbidities has not yet been unanimously clarified. This work comprises aliterature search and analysis of the pertinent retrospective studies and case series. Structured diagnostic workup with contrast-enhanced computed tomography (CT) is essential for planning transarterial embolization (TAE). Contrast agent extravasation, the signal flare phenomenon, and the hematocrit effect are all indicative of active bleeding or an anticoagulant-related hematoma. For TAE, coils, particles, and liquid embolic agents can be used alone or in combination. Aback door/front door embolization should be strived for. Transarterial embolization is the method of choice for managing anticoagulant-related localized or diffuse spontaneous soft tissue bleeding in the context of hemodynamic stabilization of the patient.

Liquid chromatography-mass spectrometric method for the simultaneous analysis of branched-chain amino acids and their ketoacids from dried blood spot as secondary analytes for the detection of maple syrup urine disease

BackgroundMaple syrup urine disease (MSUD) is an aminoacidopathy caused by a defective branched-chain alpha-ketoacid dehydrogenase complex, leading to the accumulation of branched-chain amino acids (BCAAs) and their respective keto acids (BCKAs). A comprehensive test was developed to measure BCAAs and BCKAs using LC-MS from dried blood spot (DBS) samples for the diagnosis and prevention of MSUD in newborns and infants. MethodsAnalytes were extracted from DBS using a methanol:0.1 % v/v formic acid solution (75:25) containing internal standards and analyzed on a Luna PFP column (150 mm × 4.6 mm, 3 µm) at a flow rate of 0.3 mL/min. The method was validated for linearity, accuracy, precision, recovery, carry-over, matrix effect, hematocrit, blood volume, and punch position effects. Biomarker stability in the matrix and stock solution was assessed. Correlation with the plasma method was determined using Pearson’s correlation coefficient and Bland-Altman analysis. The method established reference ranges for the Udupi district population in South India. ResultsThe method demonstrated linearity (r2 > 0.99), with a lower limit of detection at 2 µM (BCAA) and 1 µM (BCKA), and acceptable recovery of QC samples. Hematocrit, blood volume, punch position, and storage condition effects were within acceptable limits. Correlation and Bland-Altman analysis showed strong interconvertibility between plasma and DBS assays. Reference ranges for leucine, isoleucine, valine, KIC, KIV, and KMV were established. ConclusionThe developed DBS method, requiring no derivatization and involving simple sample preparation with short run times, is a cost-effective and reliable approach for the confirmatory diagnosis of MSUD.

Personalization of pharmacotherapy with sirolimus based on volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients-from LC-MS/MS method validation to clinical application.

The benefits of pharmacotherapy with sirolimus (SIR) in pediatric transplant recipients are well established. Traditionally, whole blood samples have been used to measure SIR concentrations. Volumetric Absorptive Microsampling (VAMS) is an alternative sampling strategy suitable for Therapeutic Drug Monitoring (TDM). In this study, we developed and validated two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for determining SIR concentrations in whole blood (WB) and capillary whole blood samples collected using a VAMS-Mitra™ device. We used protein precipitation during WB sample preparation and dispersive liquid-liquid microextraction (DLLME) with methyl tert-butyl ether for VAMS sample preparation to optimise the analyte extraction process. The described validation protocols were cross-validated, confirming the equivalence of the whole-blood and VAMS-based methods. Furthermore, the developed methods were evaluated in two three-level rounds of an external proficiency-testing scheme. The analytical methods were successfully validated within the calibration range of SIR (0.5-60 ng/ml). The validation parameters met the European Medicines Agency (EMA) and the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDM&CT) acceptance criteria. No hematocrit (tested in the range of 24.3-64.1%), matrix, or carry-over effects were observed. Cross-validation confirmed the interchangeability between VAMS-LC-MS/MS and WB-LC-MS/MS methods. The developed methods were successfully implemented for SIR determination in 140 clinical samples (70 each of WB and VAMS) from pediatric renal transplant recipients, demonstrating their practicality and reliability. The VAMS-based method has been rigorously tested and is clinically equivalent to the reference WB-LC-MS/MS method. Additionally, clinical validation confirmed the utility of the presented methods for TDM of the SIR in the pediatric population after renal transplantation.

Simultaneous influences of hematocrit in the erythrocyte medium on erythrocyte aggregation and sedimentation: a kinetic study by a laser scattering technique

The erythrocyte aggregation is an important physiological phenomenon in the circulation of blood. It is a basic characteristic of normal blood that plays a major role in the cardiovascular system, especially in the microcirculation. This study explained the kinetics of single cells rouleaux formation one- dimensional aggregate and three- dimensional aggregate, during simultaneous, and the effect of hematocrit on the process of aggregation and sedimentation. The present study was done on forty one healthy subjects. Laser light is passed through a well mixed sample of blood and the forward scattered light intensities recorded continuously. The samples were prepared with different hematocrit, (10%, 15%, 20%, and 25%). Increasing the hematocrit, (10%, 15%, 20%, and 25%) had significantly decreased the rate of rouleaux formation (P&lt; 0.005) but increase in the rate of one and three dimensional aggregate formation. On the other hand the sedimentation rate is decreased significantly (P

Efficient Green Solvent Extraction and Bioassay Studies of Favipiravir in Rat Dried Blood Spots Using Ionic Liquid-Based Dispersive Liquid-Liquid Microextraction (IL-DLLME) Coupled with HPLC-PDA: Application to Bioequivalence Studies

Background: Favipiravir is an antiviral drug having pyrazine group moiety. It is a reliable and an efficient green solvent, and a highly recoverable bio-sampling method is required for it. It is widely used in COVID-19 treatment for the prevention of the spread of viral infections in the body Objective: This proposed green solvent (ionic liquid)-based bioassay study aimed to quantify favipiravir in rat-dried blood spots using Dispersive Liquid-liquid Microextraction (IL-DLLME). Methods: The proposed bioassay separation was achieved through an isocratic elution mode using a hybrid silica-based ODS column (250 × 4.6 mm; 5 μm), a column temperature of 25°C, an injection volume of 10 μL, a flow rate of 1.0 mL/min, and a detector wavelength set at 310 nm. The run time was less than 10 minutes. Mobile phase was delivered with acetonitrile, methanol, and 10 mM Na2HPO4 at pH 4.0 ± 0.5, (15: 20: 65, v/v/v). In a microtube, 50 mL of Ionic Liquid (IL), 500 μL of disperser ACN, 50 μL of 10% NaCl, and IS (20 ng mL-1) were added to perform the Dried Blood Spot (DBS) sample extraction methodology. The advantages of the proposed methodology have been found to include minimum hematocrit effect and an adequate blood volume for testing, easy transportation, and significant extraction recovery. The sample analysis has been carried out using HPLC-PDA with oseltamivir used as an internal standard. Results: We have investigated the important variables, i.e., salt concentration (10% NaCl, analyte recovery being higher) and disperser solvent [50μL of BMIHP plus 500 μL of ACN (v/v) yielding the highest recovery], in the extraction process. Extraction Recovery (ER) and Enrichment Factor (EF) were also evaluated using the IL-DLLME method. The calibration curve examined a range of 0.5-150 μg/mL, with a lower Limit of Quantification (LOQ) being 0.5 μg/mL in QC as well as calibration samples, respectively. Conclusion: Significant bioanalytical validation has been performed and all the parameters have been evaluated systematically as per bioanalytical method validation protocols, i.e., US FDA- 2018 guidelines. The following analytical parameters have been covered: standard curve, limit of quantification, range, recovery, stability, accuracy, precision, sensitivity, ER, EF, and selectivity. The developed bioassay method has been successfully applied in the pharmacokinetic studies of rats and successfully applied in bulk drugs.

Effects of hematocrit and non-Newtonian blood rheology on pulsatile wall shear stress distributions in vascular anomalies: A multiple relaxation time lattice Boltzmann approach

In the present study, we investigate the flow dynamics of non-Newtonian blood, focusing on the distribution of wall shear stress (WSS) and hematocrit levels, which is the volume percentage of red blood cells in whole blood. We analyze these factors under pulsatile conditions, in vascular anomalies such as stent channels and intracranial aneurysms. To achieve this, a three-dimensional computational approach based on the lattice Boltzmann method (LBM) with a multiple relaxation time (MRT) collision operator is employed. To represent the blood's shear-thinning properties, we developed a constitutive model inspired by the Carreau–Yasuda model. This model considers the variability in blood viscosity with shear rate correlated with hematocrit levels based on experimental data documented in the literature. The accuracy of the employed MRT-LBM is demonstrated by the consistency of results with analytical solutions for steady state and experimental data for pulsatile WSS distributions in non-Newtonian and Newtonian fluids. Results indicate that, in areas narrowed by stenosis or expanded by aneurysms, hematocrit levels affect flow dynamics. Higher hematocrit levels intensify pulsatile flow through stenotic regions, increasing WSS cyclic variations. We derived a density distribution function to demonstrate how shear rates vary in vascular anomalies, revealing blood viscosity changes and non-Newtonian properties. These properties complicate flow patterns, resulting in non-linear WSS distributions, which are essential for understanding endothelial cell reactions and disease pathways. Pulsatile blood flow and altered rheological properties due to increased hematocrit affect saccular aneurysm fluid dynamics over time and space, causing vorticities to change shape, size, and intensity.

Impact of hematocrit levels on the accuracy of specific blood glucose meters: Ahospital-based study.

Blood glucose meters are commonly used at the bedside, but most of the meters used in Hung Vuong Hospital (Ho Chi Minh City, Vietnam) are built for self-monitoring and might not be suitable for determining glucose levels in patients. In this study, we aimed to validate the performance of six frequently used meters in our hospital using the Clinical & Laboratory Standards Institute (CLSI) standard, and investigate the hematocrit impact on the accuracy of these meters. A total of 135 pregnant women who underwent a 75-g oral glucose tolerance test consented to participate in the study at Hung Vuong Hospital. Whole blood glucose levels were measured in duplicate using meters, and hematocrit levels were measured using an Alinity h-series analyzer. Within 5 min, plasma glucose levels were measured twice in a row using the Cobas c502 reference analyzer. For accuracy and precision, the hematocrit effect was assed using CLSI POCT12-A3. Out of six evaluated meters, three meters qualified. For CLSI criterion at glucose concentration of 5.55 mmol/L, Accu-Chek Inform II, Accu-Chek Performa and OneTouch VerioVue achieved 97.31%, 98.08% and 99.62%, respectively. For CLSI criterion at 4.17 mmol/L, these three achieved 100%. Accu-Chek Inform II and Accu-Chek Performa showed an inverse correlation between glucose level and hematocrit with slopes of -0.500 (95% confidence interval -0.678 to -0.322) and -0.396 (95% confidence interval -0.569 to -0.224), whereas OneTouch VerioVue was not affected by hematocrit, with a slope of 0.207 (95% confidence interval -0.026 to 0.440). Blood glucose meters' measurements can be affected by hematocrit, and might provide readings not within an acceptable bias. Medical organizations need to verify or validate before using on patients.

Accuracy of FreeStyle Libre flash glucose monitoring in patients with type 2 diabetes who migrated from highlands to plains

BACKGROUND The FreeStyle Libre flash glucose monitoring (FGM) system entered the Chinese market in 2017 to complement the self-monitoring of blood glucose. Due to its increased usage in clinics, the number of studies investigating its accuracy has increased. However, its accuracy has not been investigated in highland popu-lations in China. AIM To evaluate measurements recorded using the FreeStyle Libre FGM system compared with capillary blood glucose measured using the enzyme electrode method in patients with type 2 diabetes (T2D) who had migrated within 3 mo from highlands to plains. METHODS Overall, 68 patients with T2D, selected from those who had recently migrated from highlands to plains (within 3 mo), were hospitalized at the Department of Endocrinology from August to October 2017 and underwent continuous glucose monitoring (CGM) with the FreeStyle Libre FGM system for 14 d. Throughout the study period, fingertip capillary blood glucose was measured daily using the enzyme electrode method (Super GL, China), and blood glucose levels were read from the scanning probe during fasting and 2 h after all three meals. Moreover, the time interval between reading the data from the scanning probe and collecting fingertip capillary blood was controlled to &lt; 5 min. The accuracy of the FGM system was evaluated according to the CGM guidelines. Subsequently, the factors influencing the mean absolute relative difference (MARD) of this system were analyzed by a multiple linear regression method. RESULTS Pearson’s correlation analysis showed that the fingertip and scanned glucose levels were positively correlated (R = 0.86, P = 0.00). The aggregated MARD of scanned glucose was 14.28 ± 13.40%. Parker's error analysis showed that 99.30% of the data pairs were located in areas A and B. According to the probe wear time of the FreeStyle Libre FGM system, MARD1 d and MARD2–14 d were 16.55% and 14.35%, respectively (t = 1.23, P = 0.22). Multiple stepwise regression analysis showed that MARD did not correlate with blood glucose when the largest amplitude of glycemic excursion (LAGE) was &lt; 5.80 mmol/L but negatively correlated with blood glucose when the LAGE was ≥ 5.80 mmol/L. CONCLUSION The FreeStyle Libre FGM system has good accuracy in patients with T2D who had recently migrated from highlands to plains. This system might be ideal for avoiding the effects of high hematocrit on blood glucose monitoring in populations that recently migrated to plains. MARD is mainly influenced by glucose levels and fluctuations, and the accuracy of the system is higher when the blood glucose fluctuation is small. In case of higher blood glucose level fluctuations, deviation in the scanned glucose levels is the highest at extremely low blood glucose levels.

Can Angiography Be Planned Based on Specific CT Signs of Ongoing Soft Tissue Bleeding in COVID-19 Patients Receiving Anticoagulants?

Background. Anticoagulant therapy is recommended as basic medical treatment for COVID-19 patients with risk factors. At the same time, it is accompanied by a large number of hemorrhagic complications. Massive bleeding into abdominal and chest walls soft tissues as well as retroperitoneal space significantly worsens the course of underlying disease.Objective: to assess the prognostic value of specific CT signs and their correlation with angiography data for the diagnosis of ongoing bleeding into soft tissues in COVID-19 patients.Material and methods. A retrospective study was conducted, in which multi-slice computed tomography (MSCT) and angiography data were assessed and compared in 60 COVID-19 patients with spontaneous bleeding into chest and abdominal wall soft tissues as well as retroperitoneal space. A retrospective analysis of MSCT was carried out to identify symptoms specific to coagulopathic bleeding into soft tissues: contrast agent extravasation, hematocrit effect, and signal flare phenomenon.Results. Contrast agent extravasation identified by MSCT was confirmed in 57 (95%) patients. More often (39 (68.4%) patients), extravasation was detected in MSCT arterial phase. In MSCT venous phase it was noted in 17 (29.8%) cases, in delayed phase – in 1 (1.8%) patient. With native MSCT, hematoma contained clots and fluid level (hematocrit effect) in 43 (71.7%) patients, in 17 (28.3%) patients it had a homogeneous structure. With intravenous contrast, in 39 (90.7%) cases with fluid level the signal flare phenomenon was established, which was more often detected in the arterial phase (33 (84.6%) cases). Extravasation was detected on angiography in 27 (45%) patients; they underwent therapeutic embolization. In another 15 patients preventive embolization was performed. When extravasation into hematoma without fluid level on MSCT (13 cases), extravasation on angiography was detected in 4 (30.8%) patients. In the presence of extravasation into hematoma with fluid level, but without signal flare phenomenon (4 patients) it was noted on angiography in 1 case. In the presence of all three signs (extravasation, hematocrit effect, and signal flare phenomenon – 39 patients), the incidence of extravasation on angiography was the highest – 56.4% (p = 0.028), and patients more often required therapeutic embolization.Conclusion. Detection of extravasation, fluid level and signal flare phenomenon on MSCT in the structure of spontaneous hematoma in COVID-19 patients corresponded to ongoing bleeding and more often required embolization than in isolated extravasation.

Pre-operative anaemia-does it influence post-operative outcomes after isolated coronary artery bypass grafting?

Preoperative anaemia is prevalent in a number of patients undergoing coronary artery bypass grafting. Studies provide conflicting results due to several reasons including variation in the threshold of haematocrit used to define anaemia. We aimed to assess the independent effect of preoperative anaemia on outcomes in patients undergoing off pump coronary artery bypass grafting (OPCAB). In this retrospective study, patients with a hemoglobin level less than 11g/dl (haematocrit <33%) were considered to have moderate-to-severe anaemia as per the recommendations of the World Health Organization. Association between haematocrit <33% and mortality as well as adverse post-operative outcomes was assessed. Multivariable logistic regression (MLR) was carried out to assess the independent effect of haematocrit<33% on 30-day mortality and other outcomes. The study included 4957 consecutive patients undergoing isolated OPCAB surgery between 2015 and 2020. Out of 4957, 635 (12.8%) had haematocrit <33% and 4322 (81.2%) had haematocrit ≥33%. Patients with haematocrit < 33% had a 30-day mortality of 13 (2%) compared to 38 (0.9%) in patients without anaemia and had a greater requirement for blood transfusion (p<0.0001). It was also associated with an increased incidence of renal failure (p<0.0001), tracheostomy (p=.0.012) and risk of re-intubation (p=0.006). On multiple linear regression (MLR), haematocrit < 33% was not an independent predictor of 30-day mortality odds ratio (OR) 1.47, 95% confidence interval (CI) 0.745-2.917; p=0.26. It was however an important independent risk factor for blood transfusion (OR 1.80, 95% CI 1.29-2.50, p<0.001) and renal failure (OR 3.06, 95% CI 1.338-7.012, p=0.008). The receiver operating characteristic (ROC)-area under the curve (AUC) was 0.63 suggesting moderate discriminatory value of haematocrit < 33% for 30-day mortality. Haematocrit < 33% is an important risk factor for adverse outcomes following isolated, primary, elective OPCAB. The online version contains supplementary material available at 10.1007/s12055-024-01746-1.

Development and validation of ivermectin quantification method in volumetric absorptive microsampling using liquid chromatography-tandem mass spectrometry

BackgroundIvermectin is a broad-spectrum anthelmintic used to control onchocerciasis from nematode parasites. As an anthelmintic, ivermectin is designed to have high levels in the gastrointestinal tract, so that the systemic intake is relatively low. Due to the very small concentration of ivermectin, a selective and sensitive approach is needed for the analysis of ivermectin in blood. Several methods have been developed using plasma and Dried Blood Spots, but there are still shortcomings due to hematocrit effects. Therefore, this study was conducted to establish a validated ivermectin analysis method with doramectin as the internal standard in using Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry. MethodsMass spectrometry equipped with triple quadrupole and positive electrospray ionization mode was used to conduct the analysis. For the biological matrix, whole blood was used by Volumetric Absorptive Microsampling and extracted using a protein precipitation technique with a combination of acetonitrile and methanol (1:1). VAMS has some advantages such as not being affected by hematocrit, requires a small and fixed volume of sample, also a more efficient sampling process. ResultsThe optimum conditions were achieved with an Acquity® UPLC BEH C18 column (1,7 μm; 2.1 × 100 mm); extracted-flow rate was 0,2 mL/min; mobile phase was 5 mM ammonium formate pH 3.00 and acetonitrile (10:90) with isocratic elution. Multiple Reaction Monitoring (MRM) detection by m/z values was 892.41 > 569.5 for ivermectin and 916,41 > 331,35 for doramectin. ConclusionThe method has been appropriately validated in compliance with the 2018 guidelines laid out by the US Food and Drug Administration. Resulting the minimum detection (LLOQ) was 1 ng/mL with a linear concentration range spanning from 1 to 150 ng/mL.

Advances in the use of dried blood spots on filter paper to monitor kidney disease

Patients with kidney disease require frequent blood tests to monitor their kidney function, which is particularly difficult for young children and the elderly. For these people, the standard method is to evaluate serum creatinine or cystatin C or drug levels through venous sampling, but more recently, evaluation using dried blood spots has been used. This narrative review reports information from the literature on the use of dried blood spots to quantify the main markers used to detect kidney diseases. The ScienceDirect and PubMed databases were searched using the keywords: “dried blood on filter paper,” “markers of renal function,” “renal function,” “creatinine,” “cystatin C,” “urea,” “iohexol,” and “iotalamate.” Studies using animal samples were excluded, and only relevant articles in English or Spanish were considered. Creatinine was the most assessed biomarker in studies using dried blood spots to monitor kidney function, showing good performance in samples whose hematocrit levels were within normal reference values. According to the included studies, dried blood spots are a practical monitoring alternative for kidney disease. Validation parameters, such as sample and card type, volume, storage, internal patterns, and the effects of hematocrit are crucial to improving the reliability of these results.

Influence of Hematocrit Level and Integrin αIIbβIII Function on vWF-Mediated Platelet Adhesion and Shear-Induced Platelet Aggregation in a Sudden Expansion.

Shear-mediated thrombosis is a clinically relevant phenomenon that underlies excessive arterial thrombosis and device-induced thrombosis. Red blood cells are known to mechanically contribute to physiological hemostasis through margination of platelets and vWF, facilitating the unfurling of vWF multimers, and increasing the fraction of thrombus-contacting platelets. Shear also plays a role in this phenomenon, increasing both the degree of margination and the near-wall forces experienced by vWF and platelets leading to unfurling and activation. Despite this, the contribution of red blood cells in shear-induced platelet aggregation has not been fully investigated-specifically the effect of elevated hematocrit has not yet been demonstrated. Here, a microfluidic model of a sudden expansion is presented as a platform for investigating platelet adhesion at hematocrits ranging from 0 to 60% and shear rates ranging from 1000 to 10,000 s-1. The sudden expansion geometry models nonphysiological flow separation characteristic to mechanical circulatory support devices, and the validatory framework of the FDA benchmark nozzle. PDMS microchannels were fabricated and coated with human collagen. Platelets were fluorescently tagged, and blood was reconstituted at variable hematocrit prior to perfusion experiments. Integrin function of selected blood samples was inhibited by a blocking antibody, and platelet adhesion and aggregation over the course of perfusion was monitored. Increasing shear rates at physiological and elevated hematocrit levels facilitate robust platelet adhesion and formation of large aggregates. Shear-induced platelet aggregation is demonstrated to be dependent on both αIIbβIII function and the presence of red blood cells. Inhibition of αIIbβIII results in an 86.4% reduction in overall platelet adhesion and an 85.7% reduction in thrombus size at 20-60% hematocrit. Hematocrit levels of 20% are inadequate for effective platelet margination and subsequent vWF tethering, resulting in notable decreases in platelet adhesion at 5000 and 10,000 s-1 compared to 40% and 60%. Inhibition of αIIbβIII triggered dramatic reductions in overall thrombus coverage and large aggregate formation. Stability of platelets tethered by vWF are demonstrated to be αIIbβIII-dependent, as adhesion of single platelets treated with A2A9, an anti-αIIbβIII blocking antibody, is transient and did not lead to sustained thrombus formation. This study highlights driving factors in vWF-mediated platelet adhesion that are relevant to clinical suppression of shear-induced thrombosis and in vitro assays of platelet adhesion. Primarily, increasing hematocrit promotes platelet margination, permitting shear-induced platelet aggregation through αIIbβIII-mediated adhesion at supraphysiological shear rates. The online version contains supplementary material available at 10.1007/s12195-024-00796-0.

Hematocrit-Independent Sampling Enables White Blood Cell Counts from Patterned Dried Blood Spot Cards.

The accurate and efficient measurement of white blood cell (WBC) counts is vital for monitoring general patient health and can aid in the diagnosis of a range of possible infections or diseases. Even with their importance universally acknowledged, access to WBC counts is largely limited to those with access to phlebotomists and centralized clinical laboratories, which house the instruments that perform the tests. As a result, large populations of people (e.g., those that are home-bound or live in remote locations) lack facile access to testing. Dried blood spot (DBS) cards are often used to bridge these gaps in access to testing by offering the ability to collect blood at home for ambient shipping to laboratories. However, it is well understood that these cards, which are prepared from cellulose cardstocks without further modification, suffer from variabilities in accuracy and precision due to uncontrolled sample spreading and hematocrit effects, which have hindered their use to determine WBC counts. In this paper, we present a method to obtain an accurate WBC count using a patterned dried blood spot (pDBS) card, which comprises collection zones that meter volumes of dried blood. Using an input volume of 75 μL of whole blood, we demonstrate that, unlike the gold standard DBS card (Whatman 903), our pDBS design allows for the collection of replicate zones containing a reproducible, average volume of dried blood (12.1 μL, 7.8% CV) over the range of hematocrits from 25 to 55%. We then used qPCR to quantify the 18S rRNA gene to determine WBC counts from the volumes of blood that are metered in pDBS zones. We observe that WBC counts generated from our method are comparable to those measured with a HemoCue point-of-care WBC analyzer. Our approach to using pDBS cards as a blood collection device has the potential to support at-home sampling and other patient populations that need WBC counts but lack access to clinical facilities.

A Validated Assay to Quantify Osimertinib and Its Metabolites, AZ5104 and AZ7550, from Microsampled Dried Blood Spots and Plasma.

Osimertinib is an oral small-molecule tyrosine kinase receptor inhibitor used to treat non-small cell lung cancer (NSCLC) with a sensitizing epidermal growth factor receptor mutation. Patients may experience drug toxicity and require dose deescalation. The study aimed to quantitate osimertinib and its 2 active metabolites, AZ5104 and AZ7550, in microsampled dried blood spots (DBS) collected from patients with NSCLC using a hemaPEN device and compare them with plasma drug levels. A 6-min ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated using plasma and DBS. The accuracy, selectivity, matrix effect, recovery, and stability were assessed using bioanalytical validation criteria. The hematocrit effect was investigated in DBS. Drug levels were measured in 15 patients with NSCLC, and the Bland-Altman method was used to compare measurements between plasma and DBS. The validated assay determined accurate and precise quantities, respectively, for osimertinib in both plasma (93.2%-99.3%; 0.2%-2.3%) and DBS (96.7%-99.6%; 0.5%-10.3%) over a concentration of 1-729 ng/mL. The osimertinib metabolites, AZ5104 and AZ7550, were similarly validated in accordance with bioanalytical guidelines. For 30%-60% patient hematocrit, no hematocrit bias was observed with DBS for all analytes. The Bland-Altman method showed high concordance between plasma and DBS analyte levels. Stability experiments revealed that osimertinib and its metabolites were poorly stable in plasma at room temperature, whereas all analytes were stable in DBS for 10 days at room temperature. The measurement of osimertinib, AZ5104, and AZ7550 from hemaPEN microsampled DBS is a convenient and reliable approach for therapeutic drug monitoring that produces measurements consistent with plasma drug levels.

Portable platform for leukocyte extraction from blood using sheath-free microfluidic DLD.

Leukocyte count is routinely performed for diagnostic purposes and is rapidly emerging as a significant biomarker for a wide array of diseases. Additionally, leukocytes have demonstrated considerable promise in novel cell-based immunotherapies. However, the direct retrieval of leukocytes from whole blood is a significant challenge due to their low abundance compared to erythrocytes. Here, we introduce a microfluidic-based platform that isolates and recovers leukocytes from diluted whole blood in a single step. Our platform utilizes a novel, sheathless method to initially sediment and focus blood cells into a dense stream while flowing through a tubing before entering the microfluidic device. A hexagonal-shaped structure, patterned at the device's inlet, directs all the blood cells against the channel's outer walls. The focused cells are then separated based on their size using the deterministic lateral displacement (DLD) microfluidic technique. We evaluated various parameters that could influence leukocyte separation, including different focusing structures (assessed both computationally and experimentally), the orientation of the tubing-chip interface, the effects of blood sample hematocrit (dilution), and flow rate. Our device demonstrated the ability to isolate leukocytes from diluted blood with a separation efficiency of 100%, a recovery rate of 76%, and a purity of 80%, while maintaining a cell viability of 98%. The device operates for over 30 min at a flow rate of 2 μL min-1. Furthermore, we developed a handheld pressure controller to drive fluid flow, enhancing the operability of our platform outside of central laboratories and enabling near-patient testing. Our platform can be integrated with downstream cell-based assays and analytical methods that require high leukocyte purity (80%), ranging from cell counting to diagnostics and cell culture applications.

Dried blood spot analysis for the quantification of vancomycin and creatinine using liquid chromatography – tandem mass spectrometry: Method development and validation

BackgroundVancomycin is a widely used antibiotic for the treatment of gram-positive bacterial infections, especially for methicillin-resistant Staphylococcus aureus (MRSA) infections. Due to a small therapeutic range and large inter-patient variability, therapeutic drug monitoring (TDM) of vancomycin is required to minimize toxicity and maximize treatment efficacy. Venous blood sampling is mostly applied for TDM of vancomycin, although this widely used sampling method is more invasive compared to less painful alternatives, such as the dried blood spot (DBS) method, which can be performed at home. MethodWe developed an UPLC-MS/MS method for the quantification of vancomycin and creatinine in DBS. A fast sample preparation and short analysis run time of 5.2 min were applied, which makes this method highly suitable for clinical settings. Validation was performed according to international (FDA and EMA) guidelines. ResultsThe validated concentration range was found linear for creatinine from 41.8 µmol/L to 722 µmol/L and for vancomycin from 3.8 mg/L to 76.6 mg/L (r2 > 0.990) and the inaccuracies, imprecisions, hematocrit effects, and recoveries were < 15 % for both compounds. No significant carryover effect was observed. ConclusionHence, we successfully validated a quantification method for the simultaneous determination of creatinine and vancomycin in DBS.

Microfluidic viscometer using capillary pressure sensing

Blood viscosity is considered as a vital determinant of the efficiency of blood flow in blood-vessel networks. The coflowing method is considered as a promising technique for measuring blood viscosity. However, it requires two precise syringe pumps to supply two fluids (i.e., the reference fluid and blood), calibration in advance, and long waiting time for securing steady blood flow. To solve these problems, a single syringe pump is adopted to supply blood into a microfluidic device without requiring a reference fluid. Two key parameters—fluidic resistance and compliance coefficient—are suggested and obtained by analyzing the fluid velocities in a microfluidic channel and calculating the air pressure in the air compliance unit. Using a discrete fluidic circuit model, the pressure difference is analytically derived and utilized as the nonlinear regression formula. The two key parameters are then obtained through nonlinear regression analysis. According to experimental results, the air cavity and flow rate contribute to increasing the compliance coefficient. The fluidic resistance increases significantly at higher concentrations of glycerin solution ranging from 20% to 50%. The proposed method underestimates the values by approximately 27.5% compared with the previous method. Finally, the proposed method is adopted to detect the effects of hematocrit and red blood cell sedimentation in the driving syringe based on two vital parameters. Regarding the fluidic resistance, the normalized difference between the proposed and previous methods is less than 10%. Therefore, two key parameters can be considered as effective for quantitatively monitoring the hematocrit variation in blood flow. In conclusion, from a biomechanical perspective, the proposed method is highly promising for quantifying blood flow in a microfluidic channel.