Influence Of Hematocrit Research Articles

Influence of Hematocrit and Storage Temperature on the Stability of Dried Blood Samples in Serological Analyses of Tetanus, Diphtheria, and Pertussis.

Dried blood spots (DBSs) enable the study of serological markers of various pathogens without the need to obtain serum/plasma through venipuncture. Sixty-four blood samples were prepared on Whatman™ 903 cards using specimens obtained by venipuncture to study the detection of serological markers of diphtheria, tetanus, and pertussis in DBSs, and their stability 4 months post-collection. An automated chemiluminescent immunoassay was used to investigate diphtheria, tetanus, and pertussis IgG levels from both DBSs and plasma samples. An optimal cut-off value for DBSs was calculated to improve the performance of diphtheria and tetanus serological markers in DBSs, achieving high sensitivity (95% and 98%, respectively) and specificity (91.7% and 92.3%, respectively). No protection against pertussis was found in the population studied. The correlation observed between the plasma and the DBSs processed after sample collection was high (0.967-0.970) for all antibodies studied except pertussis (0.753), both considering hematocrit before sample elution or not. The correlation between DBSs and plasma for diphtheria and tetanus remained strong following a 4-month delay in DBS processing at 4 °C (0.925-0.964) and -20 °C (0.924-0.966), with only a minor decrease observed for diphtheria at room temperature (0.889), while maintaining a strong correlation for tetanus (0.960). For pertussis, the correlation between DBSs and plasma was drastically reduced after delaying its processing for 4 months at any temperature. To summarize, hematocrit levels within the normal range do not affect the processing of DBSs in the study of serological markers of diphtheria, tetanus, and pertussis. The DBS stability for serological diagnosis of diphtheria and tetanus is adequate when samples are stored at -20 °C for a period of 4 months. The pertussis serological marker does not appear to remain stable after 4 months, even when the DBS is stored frozen at -20 °C.

Evaluating the impact of transient shear stress on platelet activation, adhesion, and aggregation with microfluidic chip technique.

When nonphysiological stenosis occurs, the transient high shear stress formed in vessels increases the risk of thrombosis and is a potential factor for cardiovascular diseases. But the platelet adhesion and aggregation behavior at nonphysiological post-stenosis and its affecting factors are not fully understood yet. In this experiment, platelet aggregation on collagen and fibrinogen at different shear stresses and different hematocrits were observed by microfluidic technology. Platelet activation (P-selectin, glycoprotein IIb/IIIa) and monocyte-platelet aggregate (MPA) levels under different shear stresses were analyzed by flow cytometry. On fibrinogen, platelets aggregate more at higher shear stress conditions. While on collagen, it becomes more difficult for platelets to form stable aggregation at higher shear stress conditions. If platelets adhere initially at low shear stress, stable platelet aggregation can be formed at subsequent high shear stress. Moreover, when the shear stress increases, platelet activity markers (P-selectin, glycoprotein IIb/IIIa and MPAs) increase significantly. Hematocrit affects the degree of platelet aggregation, and the influence of hematocrit is obvious at high shear stress. Transient high shear stress (46 ms) can effectively activate platelets. Platelet aggregation behavior was different for coated fibrinogen and collagen protein. Stable platelet adhesion at post-stenosis is more dependent on fibrinogen and platelet aggregation is stable on both fibrinogen and collagen. Hematocrit can significantly affect the formation of platelet aggregation.

Evaluation of PA990pro, an Analyzer for C-Reactive Protein Using Whole Blood: Hematocrit Correction is Required.

The rapid detection of C-reactive protein (CRP) in whole blood can provide a basis for reducing the use of antibiotics, especially for infants in whom blood collection is difficult. Whether the performance of PA990pro for CRP detection can meet clinical needs has not been studied. Between May and June 2022, 230 blood samples were collected to explore the analytical performance of the PA990pro for CRP detection. The blank check, carryover, repeatability, intermediate precision, linearity, sample stability, the influence of hematocrit (HCT)/triglyceride/bilirubin, and the trueness of the PA990pro were evaluated. Whole blood CRP test results analyzed using the PA990pro were compared to plasma CRP test results from a Hitachi 7180 biochemical analyzer, using the same samples. The blank check (≤ 0.3 mg/L), carryovers (≤ 0.05%), repeatability (≤ 7.23%), and intermediate precision (≤ 7.36%) can meet clinical needs. The linear correlation coefficients of different ranges of CRP were good (r >0.975), and the slopes were all within 0.950 - 1.050. The stability of samples was good within 72 hours whether stored at 18 - 25℃ or 2 - 8℃ (CV < 10%). With interference from triglycerides at ≤ 7 mmol/L, the deviation of CRP was < 10% and with bilirubin at ≤ 216 μmol/L, the deviation of CRP was < 10%. PA990pro has no HCT quantification function; abnormal HCT will significantly interfere with whole blood CRP results (the relative deviation in the basic experiment was up to 73.71%). We suggest that the HCT results of the patient during the same period are provided through the laboratory information system (LIS) so that a CRP correction formula (CRPcorrected = CRPmeasured*(1 - 40%)/(1 - HCTmeasured)) can be used on the LIS. After using the formula to perform the HCT correction, the results of PA990pro were well correlated with the 7180 analyzer plasma CRP detection (r > 0.975). PA990pro could pass the external quality assessment of the National Center for Clinical Laboratories. The CRP detection performance of PA990pro can meet clinical needs, but it is suggested that HCT should be corrected by the formula set in LIS. It is a simple, rapid, and cost-free approach that can be used to obtain a modified whole blood CRP test result that meets clinical needs.

Dependency of R2 and R2* relaxation on Gd‐DTPA concentration in arterial blood: Influence of hematocrit and magnetic field strength

Dynamic susceptibility contrast (DSC) MRI is clinically used to measure brain perfusion by monitoring the dynamic passage of a bolus of contrast agent through the brain. For quantitative analysis of the DSC images, the arterial input function is required. It is known that the original assumption of a linear relation between the R2 (*) relaxation and the arterial contrast agent concentration is invalid, although the exact relation is as of yet unknown. Studying this relation in vitro is time‐consuming, because of the widespread variations in field strengths, MRI sequences, contrast agents, and physiological conditions. This study aims to simulate the R2 (*) versus contrast concentration relation under varying physiological and technical conditions using an adapted version of an open‐source simulation tool. The approach was validated with previously acquired data in human whole blood at 1.5 T by means of a gradient‐echo sequence (proof‐of‐concept). Subsequently, the impact of hematocrit, field strength, and oxygen saturation on this relation was studied for both gradient‐echo and spin‐echo sequences. The results show that for both gradient‐echo and spin‐echo sequences, the relaxivity increases with hematocrit and field strength, while the hematocrit dependency was nonlinear for both types of MRI sequences. By contrast, oxygen saturation has only a minor effect. In conclusion, the simulation setup has proven to be an efficient method to rapidly calibrate and estimate the relation between R2 (*) and gadolinium concentration in whole blood. This knowledge will be useful in future clinical work to more accurately retrieve quantitative information on brain perfusion.

A new dried blood spot LC-MS/MS method for therapeutic drug monitoring of palbociclib, ribociclib, and letrozole in patients with cancer

Therapeutic drug monitoring (TDM) is strongly suggested to define the proper drug dosage to overcome inter- and intra-patient variability in drug exposure, which is typically observed with oral anticancer agents, such as palbociclib (PALBO), ribociclib (RIBO) and letrozole (LETRO), all approved for the treatment of HR+, HER2- locally advanced or metastatic breast cancer (BC). Optimal TDM implementation requires a blood sampling organization that can be hampered by limited availability of health and laboratory personnel. Dried Blood Spot (DBS) sampling is proposed to overcome such limitations. The aim of this work was the development of a new LC-MS/MS method to analyze DBS samples containing PALBO, RIBO, and LETRO. Analytes extraction from DBS was performed by adding a methanolic solution containing the corresponding internal standards. LC-MS/MS analysis was performed using a LC Nexera (Shimadzu) system coupled with an API 4000 QTrap (SCIEX) mass spectrometer. The chromatographic separation was performed on a Luna Omega Polar C18 column (Phenomenex). The method was applied to 38 clinical samples collected by finger prick. The influence of hematocrit and spot size, sample homogeneity, stability, and correlation between finger prick and venous DBS measurement were assessed. The analytical validation was performed according to EMA and FDA guidelines.The analytical range of the method was 1 to 250 ng/mL for PALBO, 40 to 10000 ng/mL for RIBO, and 2 to 500 ng/mL for LETRO, where linearity was assessed, obtaining mean coefficients of determination (R2) of 0.9979 for PALBO, 0.9980 for RIBO, and 0.9987 for LETRO). The LC-MS/MS method runtime was 6.6 min. Incurred sample reanalysis demonstrated reproducibility, as the percentage difference between the two quantifications was lower than 20% for 100% of PALBO, 81.8% of RIBO, and 90.9% of LETRO paired samples. Intra- and inter-day precision (CV (%)) was lower than 11.4% and intra- and inter-day accuracy was between 90.0 and 106.5%. DBS sample stability at room temperature was confirmed for 2.5 months. A positive correlation was observed between DBS and plasma concentrations for the 3 drugs, Lin’s concordance correlation coefficients obtained by DBS normalization applying a selected strategy were 0.958 for PALBO, 0.957 for RIBO, and 0.963 for LETRO. In conclusion, a fast, easy, and reproducible DBS LC-MS/MS method for the simultaneous quantification of palbociclib; ribociclib and letrozole was developed to be used in clinical practice.

International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing.

This guidance document has been prepared on behalf of the International Council for Standardization in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for the processing of citrated blood samples for coagulation tests in clinical laboratories in all regions of the world. The following areas are included in this document: Sample transport including use of pneumatic tubes systems; clots in citrated samples; centrifugation; primary tube storage and stability; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport, storage and processing; preanalytical variables for platelet function testing. The following areas are excluded from this document, but are included in an associated ICSH document addressing collection of samples for coagulation tests in clinical laboratories; ordering tests; sample collection tube and anticoagulant; preparation of the patient; sample collection device; venous stasis before sample collection; order of draw when different sample types are collected; sample labelling; blood-to-anticoagulant ratio (tube filling); influence of haematocrit. The recommendations are based on published data in peer-reviewed literature and expert opinion.

International Council for Standardisation in Haematology (ICSH) recommendations for collection of blood samples for coagulation testing

This guidance document has been prepared on behalf of the International Council for Standardisation in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for collection of blood samples for coagulation tests in clinical laboratories throughout the world. The following processes will be covered: ordering tests, sample collection tube and anticoagulant, patient preparation, sample collection device, venous stasis before sample collection, order of draw when different sample types need to be collected, sample labelling, blood-to-anticoagulant ratio (tube filling) and influence of haematocrit. The following areas are excluded from this document, but are included in an associated ICSH document addressing processing of samples for coagulation tests in clinical laboratories: sample transport and primary tube sample stability; centrifugation; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport and storage; and preanalytical variables for platelet function testing. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Quantification of ribociclib in dried blood spots by LC–MS/MS: Method development and clinical validation

A reliable, specific, selective and robust liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the determination of ribociclib in both dried blood spot (DBS) samples and potassium EDTA plasma. DBS samples were obtained simultaneously with a plasma sample in advanced breast cancer patients treated with ribociclib. A 6 mm disk from the central part of the dried blood spot sample was punched, followed by extraction of ribociclib using liquid-liquid extraction spiked with ribociclib-d6 as internal standard. Concentrations of ribociclib in DBS samples were correlated with corresponding plasma concentrations. From the blood sample also hematocrit was determined. The method was validated for selectivity, sensitivity, precision, lower limit of detection, linearity, stability and accuracy according to the food and drug administration (FDA) guideline. The within- and between-run precisions were ≤10.6 and ≤1.07 %, respectively; while the average accuracy ranged from 100 to 103 %. The influence of hematocrit on validation parameters was tested in the range of 0.20 – 0.40 L/L. No influence of hematocrit on validation parameters was observed. Regression analysis and a Bland-Altman plot indicated correlation between the results obtained from DBS and plasma samples. A strong correlation (R2 >0.97) between DBS samples and plasma concentration from 17 breast cancer patients was found. A number of 12 out of 17 processed DBS samples (71 %) fell inside the acceptable range of 20 % difference of simultaneously obtained plasma samples. The lower limit of quantification in DBS is 10.0 ng/mL and linearity was demonstrated up to 1000 ng/mL. In conclusion, the newly developed assay met the required standard for validation. The methods were used to study ribociclib disposition in patients with advanced breast cancer.

Agreement of blood glucose measured with glucose meter in arterial, central venous, and capillary samples in adult critically ill patients.

The measurement of blood glucose in critically ill patients is still performed in many ICUs with glucose meters and capillary samples. Several prevalent factors in these patients affect the accuracy of the results and should be interpreted with caution. A weak recommendation from the Surviving Sepsis Campaign (SSC) suggests the use of arterial blood rather than capillary blood for point of care testing using glucose meters. To analyse the agreement between arterial, central venous, and capillary blood samples of glucose values measured by glucose meter in critically ill patients and study potential confounding factors. Prospective cross-sectional study in a general intensive care unit (ICU). Patients needing insulin treatment (subcutaneous or intravenous) and blood glucose control were included. Standardized collection of blood samples and measurement of glucose values with a glucometer. Agreement was studied by the Bland-Altman method and stratified analysis of disagreement-survival plots was used to study the influence of haematocrit, pH range, SOFA score, capillary refilling time, intravenous insulin infusion, and lactic acid. A total of 297 measurements from 54 patients were included. The mean arterial blood glucose was 150.42 (range 31-345 mg/dL). In the graphical analysis, there is a poor agreement both in capillary and venous central to arterial samples, but in opposite direction (underestimation of capillary and overestimation of central venous). Factors associated with a reduction in the agreement between arterial and capillary samples were elevated lactate, poor capillary refilling, and hemodynamic failure. Patients without hemodynamic compromise have an acceptable agreement with values for absolute differences of 16 mg/dL for a disagreement of 10%. In critically ill patients, the measurement of blood glucose with a glucose meter should be performed with arterial samples whenever possible. Capillary samples do not accurately estimate arterial blood glucose values in patients with shock and/or vasoactive drugs and underestimate the values in the range of hypoglycemia. Venous samples are subject to error because of potential contamination. This study adds support to the recommendation of using arterial blood rather than capillary or venous blood when using glucose meters in critically ill patients, especially in those with hemodynamic failure.

Development, validation, and application of a quantitative volumetric absorptive microsampling\u2013based method in finger prick blood by means of LC-HRMS/MS applicable for adherence monitoring of antipsychotics

Volumetric absorptive microsampling (VAMS), an emerging microsampling technique, is expected to overcome some disadvantages of dried blood spots such as volume inaccuracy and influence of hematocrit (HT). This study aimed to develop and evaluate a VAMS-based strategy for quantification of 13 frequently prescribed antipsychotics in finger prick blood within the scope of adherence monitoring to complement already-established qualitative urine analysis. The final workflow consisted of VAMS tip hydration and subsequent precipitation. Samples were analyzed by using reversed-phase ultra-high-performance liquid chromatography and Orbitrap mass spectrometry operated in parallel reaction monitoring mode. The analytical procedure was successfully validated based on international recommendations at three different HT values (20%, 40%, 60%) for most of the analytes. Selectivity and within/between-run accuracy and precision were in accordance with the recommendations in most cases. Internal standard–normalized matrix factor met recommended criteria for all analytes at HT 40%. For the HT values of 20% and 60%, only four substances did not meet the criteria. Dilution integrity was given for all substances, except for olanzapine, allowing a quantification over the whole therapeutic range of selected antipsychotics. Long-term stability in VAMS tips was tested and revealed degradation of five antipsychotic drugs after 1 week of storage at 24 °C. A proof of concept of the applicability of the method was obtained by quantification of a selection of the 13 antipsychotic drugs in VAMS tips and matched plasma samples. Results were coherent between matrices. Thus, VAMS was shown to be a promising alternative for adherence monitoring of at least the investigated antipsychotics.

Influence of Hydrodynamics and Hematocrit on Ultrasound-Induced Blood Plasmapheresis.

Acoustophoretic blood plasma separation is based on cell enrichment processes driven by acoustic radiation forces. The combined influence of hematocrit and hydrodynamics has not yet been quantified in the literature for these processes acoustically induced on blood. In this paper, we present an experimental study of blood samples exposed to ultrasonic standing waves at different hematocrit percentages and hydrodynamic conditions, in order to enlighten their individual influence on the acoustic response of the samples. The experiments were performed in a glass capillary (700 µm-square cross section) actuated by a piezoelectric ceramic at a frequency of 1.153 MHz, hosting 2D orthogonal half-wavelength resonances transverse to the channel length, with a single-pressure-node along its central axis. Different hematocrit percentages Hct = 2.25%, 4.50%, 9.00%, and 22.50%, were tested at eight flow rate conditions of Q = 0:80 µL/min. Cells were collected along the central axis driven by the acoustic radiation force, releasing plasma progressively free of cells. The study shows an optimal performance in a flow rate interval between 20 and 80 µL/min for low hematocrit percentages Hct ≤ 9.0%, which required very short times close to 10 s to achieve cell-free plasma in percentages over 90%. This study opens new lines for low-cost personalized blood diagnosis.

Hematocrit significantly confounds diffuse correlation spectroscopy measurements of blood flow.

Diffuse correlation spectroscopy (DCS) is an optical modality used to measure an index of blood flow in biological tissue. This blood flow index depends on both the red blood cell flow rate and density (i.e., hematocrit), although the functional form of hematocrit dependence is not well delineated. Herein, we develop and validate a novel tissue-simulating phantom containing hundreds of microchannels to investigate the influence of hematocrit on blood flow index. For a fixed flow rate, we demonstrate a significant inverse relationship between hematocrit and blood flow index that must be accounted for to accurately estimate blood flow under anemic conditions.

Development and validation of LC-MS/MS method for imatinib and norimatinib monitoring by finger-prick DBS in gastrointestinal stromal tumor patients.

The introduction of imatinib, an oral tyrosine kinase inhibitor, as first-line standard therapy in patients with unresectable, metastatic, or recurrent gastro-intestinal stromal tumor (GIST), strongly improved their treatment outcomes. However, therapeutic drug monitoring (TDM) is recommended for this drug due to the large inter-individual variability in plasma concentration when standard dose is administered. A Cmin higher than 760 ng/mL was associated with a longer progression free survival. Thus, a LC-MS/MS method has been developed and fully validated to quantify imatinib and its active metabolite, norimatinib, in finger-prick dried blood spot (DBS). The influence of hematocrit, sample homogeneity, and spot size and the correlation between finger-prick and venous DBS measurements were also assessed. The method showed a good linearity (R2 > 0,996) between 50-7500 ng/mL for imatinib and 10-1500 ng/mL for norimatinib. Analytes were extracted from DBS samples by simply adding to 3 mm-discs 150 μL of acidified methanol containing IMA-D8. The collected extract was then injected on a LC Nexera system in-house configured for the on-line cleanup, coupled with an API-4000 QT. The chromatographic separation was conducted on a Synergi Fusion-RP column (4 μm, 2x50 mm) while the trapping column was a POROS R1/20 (20 μm, 2x30 mm). The total run time was 8.5 min. DBSs stored at room temperature in plastic envelopes containing a silica-gel drying bag were stable up to 16 months. The proposed method was applied to 67 clinical samples, showing a good correlation between patients' finger-prick DBS and plasma concentrations, measured by the reference LC-MS/MS method, internally validated. Imatinib and norimatinib concentrations found in finger-prick DBS were adjusted by hematocrit or by an experimental correction factor to estimate the corresponding plasma measurements. At the best of our knowledge, the proposed LC-MS/MS method is the first analytical assay to measure imatinib and norimatinib in DBS samples.

Identification of the factors affecting the consistency of DBS formation via experimental design and image processing methodology

In the present study we aimed at reaching a procedure for control of factors that have a potential to contribute to bioanalytical method bias and consistent dried blood spot (DBS) formation. The most influencing factors were identified via experimental design and image processing methodology. The effects of five qualitative factors - temperature of blood samples, type of pipettes, pipetting technique, age of blood samples and analysts were investigated by multilevel categorical D-optimal design. Due to well-known influence of hematocrit (Hct) level on rheological characteristic of blood samples, five responses were observed through the study (RSD22%Hct, RSD30%Hct, RSD39%Hct, RSD51%Hct, RSD62%Hct) for their ability to provide insights into influence of factor settings onto DBS areas consistency. The area of blood spots was determined by image processing after scanning DBS cards with four spot replicates, corresponding to particular combination of investigated factors, as defined by experimental plan. The qualitative linear mathematical models with added appropriate two-factor interactions were derived by the principle of backward elimination. It was concluded that %RSD value of DBS, for all investigated Hct levels, is completely independent of type of pipettes and age of blood samples, but can significantly be affected by blood sample temperature, pipetting technique and training of analyst. Therefore, the procedure for precise and accurate formation of DBS of uniform area, regardless the Hct value, implies samples at body/room temperature, reversed pipetting technique for rigorous delivery of a sample volume onto the card and a properly trained analyst for handling samples. Finally, a verification experiment was performed and the adequacy of the suggested procedure was confirmed. Identification of the factors affecting the consistency of DBS formation provided the evidence that this contribution to total assay bias can be successfully controlled and reduced.

Dosing recommendations based on population pharmacokinetics of tacrolimus in Mexican adult patients with kidney transplant.

The aim of this study was to perform a population pharmacokinetic analysis of tacrolimus in Mexican adult kidney transplant patients to analyse the influence of clinical and genetic covariates to propose a dosage regimen. Kidney transplant patients (>18 years old) receiving oral tacrolimus treatment were included in the current study. The population pharmacokinetic model was built using a one-compartment model and the First Order Conditional Estimation method with Interaction (FOCEI via NONMEM v.7.3.). A total of 600 tacrolimus trough blood concentrations from 52 kidney transplant patients were analysed. Tacrolimus clearances were 26, 18.8 and 12.3 L/h, for patients with genetic polymorphisms CYP3A5*1*1, *1*3 and *3*3, respectively. The influence of haematocrit was inversely related to tacrolimus clearance, following an allometric power function. Total volume of distribution was 604 L. Interindividual variability associated with tacrolimus clearance and distribution volume for the final model was 33 and 63%, respectively, with a residual error of 2.5 ng/mL. Relative bioavailability was calculated between generic formulations A (0.53) and B (1) of tacrolimus. Internal validation was performed through bootstrap analysis to evaluate the stability of the final model; external validation was performed in a new group of patients (n = 13) to estimate residual errors on basic (57.8%) and final (34.8%) models. Finally, stochastic simulations were performed to propose a dosage regimen based on haematocrit, CYP3A5 genotype and generic formulation of tacrolimus. A stable and predictive population pharmacokinetic model of tacrolimus was developed for Mexican adult kidney transplant patients; additionally, the proposed dosage regimen of tacrolimus should be prospectively validated.

The relative influence of hematocrit and red blood cell velocity on oxygen transport from capillaries to tissue.

Oxygen transport to parenchymal cells occurs mainly at the microvascular level and depends on convective RBC flux, which is proportional in an individual capillary to the product of capillary hematocrit and RBC velocity. This study investigates the relative influence of these two factors on tissue PO2 . A simple analytical model is used to quantify the respective influences of hematocrit, RBC velocity, and RBC flow on tissue oxygenation around capillaries. Predicted tissue PO2 levels are compared with a detailed computational model. Hematocrit is shown to have a larger influence on tissue PO2 than RBC velocity. The effect of RBC velocity increases with distance from the arterioles. Good agreement between analytical and numerical results is obtained, and the discrepancies are explained. Significant dependence of MTCs on RBC velocity at low hematocrit is demonstrated. For a given RBC flux in a capillary, the PO2 in the surrounding tissue increases with increasing hematocrit, as a consequence of decreasing IVR to diffusive oxygen transport from RBCs to tissue. These results contribute to understanding the effects of blood flow changes on oxygen transport, such as those that occur in functional hyperemia in the brain.

The influence of hematocrit and plasma viscosity on blood viscosity

The influence of hematocrit and plasma viscosity on blood viscosity

Evaluation of the influence of hematocrit on red blood cell deformability

The present preliminary communication is to show variation in the deformability of red blood cells of Wistar rats when changes were induced in their hematocrit

Assessment of method reliability and determination of reference intervals for rotational thromboelastometry in horses.

To assess the measurement reliability of rotational thromboelastometry (ROTEM) measurements in horses, establish reference intervals for healthy horses, and evaluate the relationship between ROTEM variables, hematologic variables, and standard coagulation tests. Prospective observational study. University teaching hospital. Fifty healthy and 10 diseased adult horses. None. Blood was sampled from 10 healthy and 10 diseased horses and samples were repeatedly analyzed to evaluate measurement reliability of various ROTEM variables. Four different ROTEM assays (ie, EXTEM, INTEM, FIBTEM, and APTEM) were run simultaneously under standardized conditions. The device-related, operator-related, and day-to-day variability for the majority of ROTEM variables was very low to low, as indicated by a coefficient of variation (CV) of < 15%. Most of test-retest variability of ROTEM variables appeared to be device-related. Blood samples from 50 clinically healthy horses were used to establish reference intervals for ROTEM variables. Multiple stepwise regression analyses identified associations of different ROTEM variables with hematocrit, total protein concentration, fibrinogen concentration, platelet count, prothrombin time, activated partial thromboplastin time, and thrombin time. ROTEM is a feasible method to evaluate coagulation in horses. Its measurement reliability is acceptable, but device-related measurement variability has to be considered. Reference intervals are presented, but the influence of hematocrit, platelet count, and fibrinogen concentration may need to be taken into account when interpreting individual test results.

Analysis of ochratoxin A in dried blood spots - Correlation between venous and finger-prick blood, the influence of hematocrit and spotted volume.

We report the improvement of a method for the detection of ochratoxin A (OTA) and its thermal degradation product 2'R-ochratoxin A in dried blood spots (DBS) by high performance liquid chromatographic (HPLC) tandem mass spectrometry (MS/MS). The DBS technique was advanced for the analysis of these two compounds in DBS with unknown amounts of blood as well as varying hematocrit values. Furthermore the comparability of venous vs. capillary blood was investigated. Human whole blood samples were spotted, dried, and extracted with a solvent consisting of acetone, acetonitrile and water for analysis by HPLC-MS/MS. Quantification was carried out by stable isotope labelled internal standards. Blood samples of volunteers (n=50) were used to further optimize and simplify the procedure. Ochratoxin A and 2'R-ochratoxin A concentrations found in the entire spots (approx. 100 μL blood) were compared with punched DBS discs of 8.8mm size containing approximately 20 μL blood. As a result the amounts of both toxins in a punched 8.8mm disc correlate well with the entire DBS. Also the use of capillary blood from finger-pricks versus venous blood was evaluated. The analyte levels correlate as well indicating that the less invasive finger-prick sampling gives also reliable results. The influence of hematocrit was investigated in a range of 25-55% according to the hematocrit in the used real blood samples (34-46% hematocrit). However no significant hematocrit effect was observed for the utilized real blood samples. Moreover different blood volumes were spotted and punched as a minimal spot size is usually recommended for accurate analysis. In this experiment finger-prick samples typically consist of about 90 μL blood. Therefore spots of 75, 100 and 125 μL blood were prepared and analyzed. Similar to the hematocrit effect, no considerable influence was observed.