Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

Background:The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing.Methods:Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated.Results:Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P = 0.006; P < 0.001).Conclusion:The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.

Background: The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing.Methods: Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated.Results: Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P = 0.006; P < 0.001).Conclusion: The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.

BackgroundReliable blood‐based biomarkers (BBBMs) are urgently needed to allow for robust, cost‐effective pre‐screening of patients with suspected Alzheimer’s disease (AD) before referral for detailed assessments. We evaluated the effects of storage time and temperature, plastic tube types, and freeze/thaw (F/T) cycles in fresh, never frozen and previously frozen whole blood (WB) and plasma. Effects of anticoagulants and tube transfer were only studied in previously frozen plasma.MethodWB samples from patients with clinically suspected AD were collected in ethylenediaminetetraacetic acid (EDTA) tubes and stored at room temperature (RT) or 4°C for ≤24hrs as WB or plasma. Separated plasma was transferred into polypropylene (PP) tubes to evaluate the effects of tube transfer and F/T cycles, or into low‐density polyethylene (PE‐LD) tubes to assess the effect of tube type. In addition to EDTA, lithium heparin (Li‐Hep) and sodium citrate (NaCit) tubes were used and effects of anticoagulants evaluated. Plasma levels of phosphorylated‐tau (pTau) 217, pTau181, amyloid‐β 1–42 (Aβ42), amyloid‐β 1–40 (Aβ40), apolipoprotein E4 (ApoE4), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NFL) were measured using automated Elecsys® prototype immunoassays (cobas e 601 analyzer; all Roche Diagnostics International Ltd).ResultFor all BBBMs, pre‐analytical effects were comparable between fresh, never frozen and previously frozen samples. All BBBMs were stable for ≤24hrs at 4°C in WB and EDTA plasma; Aβ42, Aβ40, and ApoE4 were unstable if stored for &gt;2hrs at RT. Up to five tube transfers, or two F/T cycles, and both tested plastic tube types were acceptable for all BBBMs. For all BBBMs, except Aβ42 and Aβ40, analyte levels were comparable between EDTA, Li‐Hep, and NaCit tubes. Aβ42 and Aβ40 were most sensitive to pre‐analytical sample handling and the effects could only be partially compensated by the Aβ42/Aβ40 ratio.ConclusionWe provide recommendations for an optimal handling protocol for blood collection and sample handling for analysis of BBBMs for AD based on measurement in fresh samples (Figure): WB and EDTA plasma should be stored at 4°C for ≤24hrs, while storage at RT should be avoided or limited to 2hrs maximum.

Aflatoxin B1 (AFB1) is a hepatocarcinogen produced by certain Aspergillus species growing on crops. After biotransformation in the liver, AFB1 generates several metabolites, one of which is AFB1 bound to lysine on serum albumin. AFB1-lysine (AFB1-lys) is a digest product of AFB1-albumin and is considered a biomarker of exposure to AFB1 in humans and animals. The objectives of this paper were to evaluate the performance characteristics of a new analytical method for determination of AFB1-lys levels in pig serum, heparinized and ethylenediaminetetraacetic acid (EDTA) plasma and to evaluate the interference of these anticoagulants in AFB1-lys quantification. Blank blood samples were obtained from eight crossbreed 91-day-old barrows fed AFB1-free diets. Pooled samples (n = 3) and individual samples of serum, EDTA and heparinized plasma collected from five pigs were enzymatically digested with pronase at 37°C for 4 h. AFB1-lys was isolated by solid-phase extraction and quantified by liquid chromatography coupled to tandem mass spectrometry. The analytical method was applied for determination of AFB1-lys in serum and EDTA plasma collected from five 49-day-old crossbreed barrows fed ad libitum diets containing 1.1 mg of AFB1 per kg of feed during 7 days (three animals) or 42 days (two animals). Samples of heparinized plasma were only available from animals intoxicated for 42 days. All animals had lower levels of AFB1-lys in EDTA plasma samples (24.78-37.40 ng/mL), when compared to serum (49.32-252.07 ng/mL-1) or heparinized plasma (176.81 and 264.24 ng/mL-1). EDTA did not interfere in AFB1-lys standard detection, but our findings suggest that EDTA should be avoided during blood collection since it affects the pronase activity in AFB1-albumin adduct digestion and, consequently, causes a reduction in the AFB1-lys levels. Hence, determination of AFB1-lys in serum and heparinized plasma is an approach to assess an individual's exposure of swine to AFB1.

Inappropriate blood collection potentially comprises the major pre-analytical problem for coagulation testing. Inappropriate samples are most difficult to detect when received as secondary aliquots, common for referred tests. This study aimed to identify a simple, quick and inexpensive process to help laboratories distinguish the type of sample, should there be suspicion of inappropriate collection. Samples from 15 patients [selected on the basis that four different primary tubes were available: serum, citrated plasma, ethylene diamine tetraacetic acid (EDTA) plasma, lithium-heparin plasma], were tested for common electrolytes that might substantially differ according to the type of sample. In citrated plasma, potassium, chloride, calcium and magnesium were significantly decreased compared with serum and lithium-heparin plasma, while sodium was markedly increased. In EDTA plasma, sodium and chloride were significantly decreased compared with both serum and lithium-heparin plasma, potassium was always >14 mmol/l, whereas magnesium and calcium were virtually undetectable. These data allowed development of two algorithms for differential identification of citrated plasma vs. other samples with 100% sensitivity and specificity, the former based on the sequential measurement of potassium, calcium and sodium, the latter on potassium and sodium. These simple assays can supplement classical coagulation test methods to identify most inappropriate blood collections and validate sample rejection.

Tigecycline and ciprofloxacin were employed as the model compounds to study the effect of the anticoagulant ethylenediamine tetra-acetic acid (EDTA), which is used during plasma sample preparations, on the determination of pharmacokinetic parameters. The pharmacokinetic parameters were determined in rats following intravenous infusion with blood samples collected in serum separators, with either EDTA- or heparin-coated tubes. The blood-to-plasma (B:P) partition ratio and plasma protein binding were determined in vitro in rat or human blood collected in either EDTA- or heparin-coated tubes. Drug concentrations were quantified by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) analysis. In tigecycline-treated rats drug concentrations were twofold lower in EDTA plasma, leading to a twofold lower area under plasma concentration–time curve (AUC) and twofold higher plasma clearance values as compared with those obtained from heparin plasma. No differences were noted in the pharmacokinetic parameters obtained from heparin-treated plasma versus serum. The B:P partition ratio and unbound fraction for tigecycline were significantly higher in EDTA-treated blood. When normalized to the B:P partition ratios, the tigecycline blood clearance values were identical between samples collected in EDTA- or heparin-coated tubes. Similar but smaller differences were observed for ciprofloxacin. It was concluded that EDTA might compete with tigecycline and ciprofloxacin for chelating metal ions and thus affect drug partition between blood and plasma compartments, leading to inaccurate measurement of pharmacokinetic parameters in plasma.

The reliability of plasma biomarkers of Alzheimer's disease (AD) can be compromised by protease-induced degradation. This can limit the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). In this study, we conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 h. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0 h or 24 h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA and P100 tubes, followed by storage at RT for 0 h or 24 h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improves the stability of Aβ42 and Aβ40 across all approaches. However, the Aβ42/Aβ40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aβ42 and Aβ40, and the Aβ42/40 ratio for the IP-MS assay. These findings have crucial implications for preanalytical procedures, particularly in resource-limited settings.

Widal examinaton is an agglutination test used in the serological diagnosis of typhoid fever. Widal examination consists of two methods, namely qualitative and quantitative methods. Specimens that can be used for serological tests are serum, plasma and cerebrospinal fluid. The plasma used is EDTA plasma. The purpose of this study was to determine the differences in the use of serum and EDTA plasma on the results of the quantitative method of Widal examination. This study was conducted at the Clinical Pathology Laboratory of Roemani Hospital, Semarang. This type of research is an analytical study with a cross-sectional approach conducted from October to November 2024. The samples studied were 16 serum samples and 16 EDTA plasma samples. The results of the Widal examination using serum obtained low titer results of 1/80 in 11 samples (34.4%), medium titer 1/160 in 16 samples (50%) and high titer 1/320 in 5 samples (15.7%) with a research sample of 32 (100%), while in plasma obtained low titer results of 1/80 in 26 samples (81.3%), medium titer 1/160 in 5 samples (15.7%) and high titer 1/320 in 0 samples (0%) with a research sample of 32 (100%). The results of the study showed that there were differences in the results of the Widal examination using serum and EDTA plasma on the results of the Widal examination.

Evaluation of a multiplex liquid chromatography-tandem mass spectrometry method for congenital adrenal hyperplasia in pediatric patients

EDTA-dependent pseudo thrombocytopenia (EDTA-PTCP) refers to a falsely low platelet count occurring in the presence of ethylene diamine tetra-acetic acid (EDTA) anticoagulant during blood sample collection, which results in the formation of platelet clumps in vitro. This phenomenon has significant clinical implications, including unnecessary administration of platelets. Our study aims to evaluate the efficacy of sodium citrate anticoagulant for the resolution of EDTAPTCP. This retrospective study was conducted in the haematology laboratory of Shifa International Hospital (SIH), Pakistan. Patients with pseudo thrombocytopenia (i.e. platelet count less than 150,000/ul with platelet clumps seen on peripheral smear) were included in this study if they had blood samples drawn in both EDTA and sodium citrate tubes less than 48 hours apart. Data was analyzed using IBM® SPSS Software Version 22. A total of 151 study participants were included in this study. The mean age was 48.95±20.69 years and the majority were female (52.3%). Wilcoxon signed-rank test showed that there was a statistically significant difference in platelet count measured in both tubes (Z = -3.223, p=0.001). Overall, blood samples processed in sodium citrate tubes showed lower platelet count than EDTA samples. Sodium citrate anticoagulant was able to correct EDTA-PTCP in 47 (31.1%) of the cases. Sodium citrate anticoagulant was only able to resolve one-third of our EDTA-PTCP cases. Our findings do not support the use of sodium citrate as a suitable alternative for correction of EDTA-PTCP.

Fibroblast growth factor 23 (FGF-23) is a recently discovered hormone, which plays a key role in phosphate regulation. To investigate whether FGF-23 can be determined reliably, we validated the three available FGF-23 assays. Currently, two intact FGF-23 assays (Kainos; Immutopics) and one C-terminal FGF-23 assay (Immutopics) are available. We determined intra- and inter-assay variation, linearity and matrix interference in these assays. Moreover, we compared assay results from healthy subjects with those of patient groups with expected high FGF-23 concentrations. Intra-assay variation was reasonably good in all three assays. Inter-assay variation and linearity were poor for the intact Immutopics assay but reasonable for both other assays. Immutopics assays gave best results in ethylenediaminetetraacetic acid (EDTA) plasma, while the Kainos assay showed comparable reproducibility in EDTA plasma and serum. Although the manual of the Kainos assay states that an automatic washing machine can be used, acceptable results were only found by manual washing. Patients with kidney disease and patients with hypophosphatemic osteomalacia had increased C-terminal FGF-23 concentrations compared with healthy controls. Two assays of reasonable quality are available for FGF-23, the intact FGF-23 assay (Kainos) provided proper attention is paid to the washing procedure, and the C-terminal assay (Immutopics).

BackgroundThe quality of laboratory test results depends on various factors, including sample type selection. Blood samples, such as whole blood, plasma and serum are commonly used for most clinical laboratory examinations. D-dimer parameters are frequently analysed in haematology laboratories and serve as biomarkers for coagulation activation and fibrinolysis. This study aimed to assess the impact of using different sample types on the quality of D-dimer test results.MethodAn observational analytical method was used. D-dimer examination was performed using the fluorescent lateral flow immunoassay method. The study sample consisted of 26 participants aged between 18 years old and 22 years old who had no blood disorders. Whole blood and ethylenediaminetetraacetic acid (EDTA) plasma samples were used for the examination of D-dimer levels.ResultsD-dimer levels in 26 participants using whole blood samples had a mean value of 0.23 mg/L (230 ng/mL), while plasma samples yielded a mean value of 0.14 mg/L (140 ng/mL). D-dimer levels obtained from whole blood samples were higher than plasma samples but remained within the normal range of 0 mg/L–0.5 mg/L (0 ng/mL–500 ng/mL).ConclusionThe results showed that whole blood samples were more practical than plasma samples. Nevertheless, plasma samples gave results within the normal range of D-dimer values.

Wildfires pose a major health risk for humans, wildlife, and domestic animals. We previously discovered pathophysiologic parallels between domestic cats with naturally occurring smoke inhalation and thermal burn injuries and human beings with similar injuries; these were characterized by transient myocardial thickening, cardiac troponin I elevation and formation of intracardiac thrombosis. While the underlying mechanisms remain unclear, results from murine models suggest that platelet priming and activation may contribute to a global hypercoagulable state and thrombosis. Herein, we evaluated and compared the degree of platelet activation, platelet response to physiologic agonists and levels of platelet-derived microvesicles (PDMV) in 29 cats with naturally occurring wildfire thermal injuries (WF), 21 clinically healthy cats with subclinical hypertrophic cardiomyopathy (HCM) and 11 healthy cats without HCM (CC). We also quantified and compared circulating PDMVs in WF cats to CC cats. In addition, we examined the association between thrombotic events, severity of burn injuries, myocardial changes, and the degree of platelet activation in cats exposed to wildfires. Flow cytometric detection of platelet surface P-selectin expression showed that WF cats had increased platelet response to adenosine diphosphate (ADP) and thrombin compared to the two control groups indicating the presence of primed platelets in circulation. In addition, cats in the WF group had increased circulating levels of PDMV, characterized by increased phosphatidylserine on the external leaflet. Cats in the WF group with documented intracardiac thrombosis had elevated platelet activation and platelet priming in the presence of ADP. While high dose arachidonic acid (AA) mostly resulted in platelet inhibition, persistent response to AA was noted among cats in the WF group with intracardiac thrombosis. Univariate and multiple logistic regression analyses demonstrated that increased platelet response to AA was independently associated with thrombotic events. This is the first study reporting the significant association between platelet priming and intracardiac thrombosis in domestic cats with naturally occurring wildfire-related injuries and smoke inhalation. Further studies are required to delineate additional mechanisms between inflammation and thrombosis, especially regarding platelet primers and the cyclooxygenase pathway.One Sentence SummaryPlatelet activation and shedding of platelet-derived microvesicles due to platelet priming is present following naturally occurring wildfire smoke exposure and thermal burn injuries in a population of domestic cats.

BackgroundMembrane-CD14 (mCD14) is expressed on the surface of monocytes, macrophages and polymorphonuclear neutrophil leukocytes (PMN). mCD14 acts as a co-receptor along with Toll like receptor 4 (TLR 4) and MD-2 for the detection of lipopolysaccharide (LPS). However, studies using different sample preparation methods and anticoagulants have reported different levels of mCD14 on the surface of monocytes and neutrophils. In this study, the influence of various anticoagulants and processing methods on measurement of mCD14 on monocytes and neutrophils was examined.ResultsWhole blood samples were collected in vacutainer tubes containing either sodium heparin (HEPARIN), ethylenediaminetetraacetic acid (EDTA) or sodium citrate (CITRATE). mCD14 on neutrophils and monocytes in whole blood samples or isolated cells was measured by the method of flow cytometry using fluorescein isothiocyanate (FITC)-labeled monoclonal antibody. There was a significant difference (p < 0.05) in the mean channel fluorescence intensity (MFI) of mCD14 on neutrophils in whole blood samples anticoagulated with HEPARIN (MFI = 64.77) in comparison with those in whole blood samples anticoagulated with either EDTA (MFI = 38.25) or CITRATE (MFI = 43.7). The MFI of mCD14 on monocytes in whole blood samples anticoagulted with HEPARIN (MFI = 206.90) was significantly higher than the MFI in whole blood samples anticoagulated with EDTA (MFI = 149.37) but similar to that with CITRATE (MFI = 162.55). There was no significant difference in the percentage of whole blood neutrophils or monocytes expressing mCD14 irrespective of type of anticoagulant used. However, MFI of mCD14 on monocytes was about 3.2-folds (HEPARIN), 3.9-folds (EDTA) or 3.7 folds (CITRATE) higher than those on neutrophils. Furthermore, there was no significant difference in mCD14 levels between unprocessed whole blood monocytes and monocytes in peripheral blood mononuclear cell preparation. Conversely, a highly significant difference was observed in mCD14 between unprocessed whole blood neutrophils and isolated neutrophils (p < 0.05).ConclusionFrom these results, it is suggested that sodium heparin should be the preferred anticoagulant for use in the reliable quantification of the surface expression of mCD14. Furthermore, measurement of mCD14 is best carried out in whole blood samples, both for neutrophils and monocytes.

concluding that the measurement of iron in ethylenediamine tetraacetic acid (EDTA) plasma may be used as a simple and cost effective approach for screening blood transfusion misuse in sports. Although these results are intuitively appealing in the spasmodic fight against doping, they seem at odds with the current recommendations for iron assessment in biological fluids. EDTA is an aminopolycarboxylic acid, which is widely used as an additive and anticoagulant for hematological testing. The underling principle supporting the use in laboratory diagnostics relies in its well established function as a ligand and chelating agent, mirrored by the powerful ability to sequester (i.e., chelate) calcium as well as a number of other metal ions including chromium, lead, mercury, copper, aluminium, nickel, zinc and, last but not least, iron. [2] It is also noteworthy that EDTA chelation for treatment of iron overload has been largely used in the past decades, and is still used in selected categories of patients due to its effectiveness and low toxicity. [3]