Heparin Tubes Research Articles

Ultrastructural changes in red blood cells in aortic dissection patients under extracorporeal circulation: Atomic force microscopy study.

During extracorporeal circulation in heart surgery, blood is exposed to non-physiological conditions, such as high shear stress, foreign surfaces, turbulence, and hypothermia. These factors cause damage to the red blood cells, which is manifested by immediate and delayed hemolysis or some changes in the mechanical properties of red blood cells, defined as sublethal trauma. Unfortunately, sublethal trauma is hard to detect, and there is not enough morphological evidence regarding red blood cell sublethal trauma. In this study, red blood cell sublethal trauma was observed after extracorporeal circulation by describing ultrastructural changes in red blood cell membranes using atomic force microscopy and scanning electron microscopy. Venous blood (2 mL) was collected into heparin tubes from preoperative, intraoperative and postoperative aortic dissection patients for comparison with blood from healthy patients. The red blood cell morphological study (malformations percentage, diameter, height, concavity, and roughness) was performed with scanning electron microscopy and atomic force microscopy. Scanning electron microscopy and atomic force microscopy imaging analysis revealed that the red blood cell shape changed during extracorporeal circulation and that the red blood cell malformation percentage in the postoperative group was higher than those in the preoperative and intraoperative groups. Most morphological parameters had no obvious changes, except roughness (Ra and Rq) in aortic dissection patients. Atomic force microscopy quantitative analysis indicated that the roughness of red blood cell membranes increased during extracorporeal circulation. This study demonstrates that ultrastructural morphological damage occurs to red blood cells membranes due to extracorporeal circulation in aortic dissection patients. In addition, we provided a new parameter (Ra and Rq) to evaluate red blood cell sublethal trauma.

Validation of an Enclosed Blood Collection System in a Pediatric Laboratory.

Preanalytical, analytical, and postanalytical issues are often magnified in pediatric laboratories, and traditional vacuum-based blood tubes can contribute to some of these issues. Because of this, we investigated adopting an enclosed blood collection system that can perform vacuum or gentle aspiration blood collection, eliminating syringes, transfer device, and transfer steps, as well as potentially minimizing preanalytical error in the pediatric laboratory. We embarked on a validation of this tube system, in comparison with our current collection tubes, across most in-house tests at a large pediatric hospital. Twenty adult volunteers were recruited. Blood was drawn into lithium heparin, serum, EDTA, and citrate tubes of each commercial tube type for comparison. For some tests, remnant blood from pediatric syringe draws was used when available. Samples were then processed and analyzed across all general areas of the clinical laboratory, and correlations of the results from the 2 tube systems were performed. Across 95 tests in the core laboratory and blood bank, almost all demonstrated clinically acceptable comparisons, with most R values >0.90. Only 3 of 95 tests demonstrated clinically significant differences between the tube systems. Our validation of the enclosed blood collection system demonstrated acceptable results when compared with our current collection tubes. Additionally, with some minor modifications, our automated instruments could utilize ultralow-volume tubes from the enclosed blood collection system for direct tube sampling, which is impossible using our current small-volume tubes with our main chemistry analyzer.

Order of draw practices in venous blood sampling at clinical biochemistry departments in the Danish health care system

BackgroundDeviation in blood collection procedures is a central source of preanalytical variation affecting overall analytical and diagnostic precision. The order of draw of venous sampling is suspected to affect analytical results, in particular for coagulation analysis. Here we compare the procedures in venous blood sampling among clinical biochemistry departments to assess the uniformity of order of blood draw and adherence to international guidelines in the Danish health care system. MethodsWe collected venous order of draw procedures from 49 clinical biochemistry departments at 22 public hospitals in Denmark. Procedures were compared to the international guidelines fromthe Clinical Laboratory Standards Institute (CLSI) and World Health Organization (WHO), and assessed in relation to department ISO 15189:2012 accreditation. ResultsWe observed seven different order of draw procedures related to citrate, serum, heparin, and EDTA tubes, and the use of discard tubes in relation to coagulation assays. 31 departments (63.3%) were found to adhere to CLSI and WHO guidelines. A majority of departments instructs the use of discard tubes before collection for coagulation assays in citrate tubes (44 departments; 89.8%). The citrate tube was the first sample tube to be drawn for most departments (35 departments; 75.5%); and the preferred order of non-citrate tubes was serum-heparin-EDTA (36 departments; 73.5%). Adherence to the CLSI and WHO guidelines was not associated with department ISO 15189:2012 accreditation (p = .57). ConclusionsVenous order of draw procedures is diverse at Danish clinical biochemistry departments and show moderate adherence to international guidelines.

Determination of reference intervals and comparison of venous blood gas parameters using a standard and nonstandard collection method in 51 dogs.

The aim of this study was to determine reference intervals (RI) for venous blood parameters determined with the RAPIDPoint 500 (RP500) blood gas analyzer using blood gas syringes (BGS) and to determine whether immediate analysis of venous blood collected into lithium heparin (LH) tubes can replace anaerobic blood sampling into BGS. The null hypothesis was that canine venous blood samples collected in BGS and in LH tubes are comparable. Jugular blood was collected from 51 healthy dogs into a BGS and a LH tube. The BGS was immediately analyzed followed by the LH tube. The RI were calculated from BGS results. The BGS and LH tubes results were compared using paired t-test or Wilcoxon matched-pairs signed-rank test and Bland-Altman analysis. To assess clinical relevance, the bias between BGS and LH tubes was compared with the allowable total error (TEa). Values derived from LH tubes showed no significant difference for standard bicarbonate (HCO3std), whole blood base excess (BE B), Na, K, Cl, glucose and hemoglobin (tHb). The pH, partial pressure of carbon dioxide and oxygen, actual bicarbonate, extracellular base excess, ionized Ca, anion gap and lactate were significantly (p.

A Simplified Sample Preparation Method for the Assessment of Plasma Ascorbic Acid in Clinical Settings.

Vitamin C deficiency is difficult to diagnose on the basis of clinical presentation alone and requires plasma levels for confirmation. Reference laboratories typically specify shipment of plasma on dry ice. This requirement may complicate clinic work flow and delay vitamin C measurement. Additionally, patients with vitamin C deficiency may experience unnecessary testing and increased health-care costs, as other diagnoses are often considered first. We examined an alternative, more practical shipping method. Plasma was collected from 17 healthy volunteers by use of heparin tubes with gel separators, and all tubes were centrifuged immediately to separate the plasma layer from the cells. Baseline vitamin C was measured in plasma obtained immediately after specimen collection. Remaining sample tubes were held in Styrofoam containers with cold packs for 30 h or 48 h, followed by vitamin C measurement. Additional samples were exposed to conditions that simulated harsher shipping conditions. Mean plasma vitamin C was 69.6 μmol/L (SD = 21.5 μmol/L). Vitamin C losses were 5.4% at 30 h (SD = 5.55%, P < 0.05) and 7.6% at 48 h (SD = 5.56%, P < 0.05), which is slightly more than freeze-and-thaw treatment (average loss of 1.4%, SD = 6.9%, NS). The vitamin C method had an intraday variation of 1.88%. Vigorous shaking of 2 samples for 24 h resulted in a -1.9% change in 1 sample, and a +4.1% change in another sample. Exposure of the shipping container to elevated temperature (35 °C for 30 h) did not change the internal temperature of the container. The shipping procedure uses routine sample handling, standard vacutainers, and can be replicated by health-care centers seeking to evaluate patient vitamin C status.

Standardization of Sampling for Isolation of Exosome-Like Small-Extracellular Vesicles from Peripheral Blood from Reproductive-Aged Women

Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of their cargo molecules, there is still a need for standardization of sampling and isolation of these microvesicles. We aimed to determine the best sampling method for isolation of sEVs from peripheral blood from reproductive-aged women. Material and Methods: We included samples of plasma from our biobank collected in 2014 by venipuncture in heparin tubes and stored at -80°C. We also included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at -80°C for one to two weeks prior processing. All blood samples were collected from the same nine reproductive-aged female volunteers. sEVs were isolated from plasma by ultracentrifugation and filtration and indirectly quantified using Pierce BCA Protein Assay kit. Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were performed to confirm the isolation of sEVs. Results and Discussion: TEM and NTA confirmed the isolation of sEVs. Protein concentration of short-time stored heparin samples was not statistically different from long-time stored heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1, p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2, p = 0.44). In conclusion, blood samples may be collected using heparin or EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs integrity and may be used, especially in comparative studies.

RNase H2-Dependent Polymerase Chain Reaction and Elimination of Confounders in Sample Collection, Storage, and Analysis Strengthen Evidence That microRNAs in Bovine Milk Are Bioavailable in Humans

Evidence suggests that dietary microRNAs (miRs) are bioavailable and regulate gene expression across species boundaries. Concerns were raised that the detection of dietary miRs in plasma might have been due to sample contamination or lack of assay specificity. Objectives: The objectives of this study were to assess potential confounders of plasma miR analysis and to detect miRs from bovine milk in human plasma. Potential confounders of plasma miR analysis (circadian rhythm, sample collection and storage, calibration, and erythrocyte hemolysis) were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR) by using blood from healthy adults (7 men, 6 women; aged 23-57 y). Bovine miRs were analyzed by RNase H2-dependent PCR (rhPCR) in plasma collected from a subcohort of 11 participants before and 6 h after consumption of 1.0 L of 1%-fat bovine milk. The use of heparin tubes for blood collection resulted in a complete loss of miRs. Circadian variations did not affect the concentrations of 8 select miRs. Erythrocyte hemolysis caused artifacts for some miRs if plasma absorbance at 414 nm was >0.300. The stability of plasma miRs depended greatly on the matrix in which the miRs were stored and whether the plasma was frozen before analysis. Purified miR-16, miR-200c, and cel-miR-39 were stable for ≤24 h at room temperature, whereas losses equaled ≤80% if plasma was frozen, thawed, and stored at room temperature for as little as 4 h. rhPCR distinguished between bovine and human miRs with small variations in the nucleotide sequence; plasma concentrations of Bos taurus (bta)-miR-21-5p and bta-miR-30a-5p were >100% higher 6 h after milk consumption than before milk consumption. Confounders in plasma miR analysis include the use of heparin tubes, erythrocyte hemolysis, and storage of thawed plasma at room temperature. rhPCR is a useful tool to detect dietary miRs.

Performance evaluation tests of lithium heparin tube with mechanical separator

Performance evaluation tests of lithium heparin tube with mechanical separator

Effect of age on chronic inflammation and responsiveness to bacterial and viral challenges.

To identify reliable biomarkers of age-related changes in chronic inflammation and responsiveness to bacterial and viral challenges, we evaluated endogenous and ex vivo stimulated levels of 18 inflammatory markers, using whole blood collected in EDTA and sodium heparin tubes from 41 healthy volunteers, i.e., 11 men + 10 women aged 20–35 and 10 men + 10 women aged 50–77. These studies revealed significant differences in the levels of inflammatory markers when blood was collected in EDTA versus sodium heparin and age related differences in these biomarkers were confirmed with blood collected in EDTA from 120 healthy volunteers in 3 age categories, ie, 20 men + 20 women, aged 20–35, 36–49 and 50–77. Studies with unstimulated blood samples, to measure levels of chronic inflammation, revealed a significant increase with age in IL-12p70, CRP and PGE2, consistent with the concept of “inflammaging”, and a decrease in G-CSF in both men and women. Interestingly, in response to E. coli stimulation, PGE2 levels were markedly reduced in the 50–77 year old cohort while they were increased following Herpes Simplex virus-1 (HSV-1) stimulation, along with IL-8. In addition, unlike E. coli, HSV-1 potently stimulated IFNα production, but levels were dramatically reduced in the older cohort, consistent with a reduced ability to generate an anti-viral response. We also found platelets and CD8+ T cells were reduced with age while CD4+ T cells were significantly increased, resulting in a substantially higher CD4/CD8 ratio in the older cohort. Surprisingly, however, we found that the older cohort exhibited more T cell proliferation and IFNγ production in response to anti-CD3+anti-CD28 stimulation. Importantly, there was considerable person-to-person variation in these inflammatory markers in all age groups, making possible comparisons between a person’s “inflammage” and chronological age. These assays should help to identify individuals at high risk of autoimmune disorders and cancer.

Short-term aerobic exercise does not change kidney function in students of Nnamdi Azikiwe University, Nigeria

BackgroundExercise has been known to have beneficial effects on human health. The kidneys play an important role in regulating acid-base and water-electrolyte balance disturbances induced by exercise. The objective of this study was to investigate the effect of short term aerobic exercise (volleyball training) on the kidney function of apparently healthy individuals. MethodsAn experimental study of pre-post test design was conducted involving 41 amateur volleyball players, comprising 22 males and 19 females. They were randomly divided into seven different teams. Each team trained for at least 45 minutes for four consecutive days for two weeks. Both pre-and post-exercise blood pressure (BP) was measured using an automatic blood pressure measuring device OMRON 907 (OMRON, Hoofddorp, Netherlands). Likewise, both pre- and post-exercise blood samples were collected into lithium heparin tubes and centrifuged at 3000 rpm for 10 minutes and the plasma separated into plain tubes. Electrolytes were analysed using ion selective electrode machine (SFRI 4000, Germany), urea using modified Berthelot method, creatinine using Jaffe-Slot method and uric acid using the uricase method and estimated glomerular filtration state (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) formula. ResultsThe mean levels of pre- and post-exercise systolic blood pressure, creatinine, urea, sodium, potassium, chloride, bicarbonate and eGFR did not differ significantly (p&gt;0.05). However, serum uric acid was significantly increased (p&lt;0.05), while diastolic BP significantly decreased after exercise (p&lt;0.05).ConclusionThe study showed that short-term moderate intensity aerobic exercise does not have any significant effect on the renal functions.

Effect of organic extracts of &lt;i&gt;Bunium incrassatum&lt;/i&gt; on the hematological, ovarian and uterine parameters of mature female rabbit

The objective of this study is to evaluate the impact of the organic extracts of Bunium incrassatum roots, belonging to the Apiaceae family, on some hematological parameter and histomorphological changes of the genital tract. This plant, called ‘Talghouda‘, is widely distributed in the east parts of Algeria. Phytochemical analyzes of the roots of this plant reveal the presence of two coumarins, scopoletin, scoparone Beta-sitosterol, sucrose and oleic acid. The study was achieved on mature rabbits of the local breed ( Oryctolagus cuniculus ) for 15 days. The animals were divided into five groups, which have daily administered 0, 25, 50, 100 and 200 mg/kg/day respectively doses of organic extract of Bunium incrassatum. The group with dose 0 was taken as a control. After treatment, the rabbits were sacrificed, the blood was collected in heparin tubes and their ovaries and uterus were removed and fixed in 10% formalin and then stained with hematoxylin-eosin. We found that treatment with the organic Bunium Incrassatum extract induces a significant decrease in the level of Triglyceride, Cholesterol in treated rabbits. The results of the histological study revealed a significant increase in the number of primary and secondary follicles in rabbits treated with 50 and 100 mg/kg/day doses and a decrease accompanied by an increase in atretic follicles in rabbits treated with a dose of 200 mg/kg/day, compared to the control group. The study shows that the organic extract of Bunium Incrassatum had estrogenic effects at doses of 50 and 100 mg/kg/day.

The local clinical validation of a new lithium heparin tube with a barrier: BD Vacutainer® Barricor LH Plasma tube.

IntroductionAlthough serum-providing blood tubes with a barrier are still widely used due to their significant advantages, the use of blood tubes with a barrier to provide plasma is becoming widespread. We compared 22 analytes in a BD Vacutainer® Barricor LH Plasma tube for local clinical validation of this new lithium heparin tube with a barrier.Materials and methodsSamples from 44 volunteers were collected in different tubes (Becton Dickinson and Company): Z tube without additive (reference), clot-activator tube with gel (SST), lithium heparin tube without gel (LiH), and lithium heparin tube with barrier (Barricor). Analyte concentrations in different tubes were compared with the reference tube. All tubes were also evaluated according to additional testing (different centrifugation durations, blood-sampling techniques and individual differences).ResultsAspartate aminotransferase (AST), glucose (Glc), potassium (K), lactate dehydrogenase (LD), sodium (Na), and total protein (TP) had a significant bias in Barricor (9.19%, - 3.24%, - 4.88%, 21.60%, - 0.40%, 5.03%, respectively) relative to the reference tube. There was no statistical difference between different centrifugation durations and individual differences for AST, K and LD in LiH and/or Barricor (P > 0.05). There was a significant bias for LD between LiH and Barricor in terms of blood-sampling techniques (21.2% and 12.4%, respectively).ConclusionsRecently, the use of plasma has become prominent due to some of its advantages. In this study, plasma AST, K, LD, Glc and TP levels in Barricor were clinically different in comparison to serum. The results of additional tests showed that higher levels of LD in Barricor did not result from haemolysis, and they might be related to other factors including number of platelets, cellular fragility, or functional environment.

Effects of different doses of heparin - encapsulated solution on coagulation mechanism in elderly patients with cardiac surgery

Objective: To investigate the effect of different doses of heparin-encapsulated liquid on the coagulation mechanism of elderly patients with cardiac surgery using superficial venous indwelling needle, and to select safe and effective dose of heparin-sealing fluid for elderly patients without increasing cardiac load. Methods: Select the use of superficial venous catheter in the heart of the coagulation function of normal elderly patients with 100 cases were randomly divided into two groups, respectively using heparin saline solution 5mL (containing heparin 62.5U / mL), 3mL (containing heparin 50.0U / mL) for indwelling needle tube, carry out before catheterization and disable heparin tube solution 24h prothrombin time (PTT), activated partial thromboplastin time (APTT), fibrinogen (Fg), and observe for any signs of bleeding (such as skin, mucous membrane with or without bleeding), while recording the retention time of indwelling needle. Results: There were no significant differences in PT, APTT and Fg between the two different doses of heparin tube (P> 0.05). There was no significant difference in the number of days between the two different doses of heparin sealed fluid and the retention time of indwelling needle (P <0.05). Conclusion: It is safe and reliable to use the above different dose of heparin sealed fluid in elderly patients with normal cardiac coagulation mechanism. Indwelling needle sealing with a small dose of low concentration of heparin sodium dilution is better, both to reduce the burden on the heart, save time, and will not affect the coagulation function.

Determination of edoxaban equivalent concentrations in human plasma by an automated anti-factor Xa chromogenic assay

IntroductionThis phase I, open-label, multiple-dose, two-treatment study assessed the relationship between edoxaban equivalent concentration derived from an anti-FXa assay with the summed concentration of edoxaban and its active metabolite, M-4, as assessed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This study also assessed the relationship between edoxaban plasma concentrations assessed by LC/MS/MS in sodium citrate and lithium heparin tubes. Materials and methodsHealthy volunteers were randomized to receive once-daily edoxaban 60mg or 90mg for 5days (15 participants per treatment group). Serial blood samples were collected for analysis by LC/MS/MS and by the anti-FXa assay. Edoxaban equivalent levels were assessed using a commercially available anti-FXa activity assay with an edoxaban-specific setup. Results and conclusionsThe day 5 concentration estimates were significantly correlated between the 2 assays (P<0.0001 for both edoxaban doses). The geometric least squares mean (GLSM) ratio (90% confidence interval) for edoxaban equivalent concentrations vs edoxaban + M-4 concentrations was 114.3% (108.2–120.8) for edoxaban 60mg (P<0.0001) and 113.0% (107.1–119.2) for edoxaban 90mg (P=0.0002). The GLSM ratio for edoxaban concentrations in sodium citrate vs lithium heparin tubes for 60-mg and 90-mg edoxaban doses were 82.8% (78.5–87.3) and 83.9% (79.1–89.0), respectively. In this study, an anti-FXa chromogenic assay with edoxaban-specific calibrators and controls demonstrated good accuracy in estimating edoxaban concentrations across a wide range of concentrations relative to LC/MS/MS at steady state following the administration of once-daily edoxaban for 5days.

Printed microfluidic filter for heparinized blood.

A simple lab-on-a-chip method for blood plasma separation was developed by combining stereolithographic 3D printing with inkjet printing, creating a completely sealed microfluidic device. In some approaches, one dilutes the blood sample before separation, reducing the concentration of a target analyte and increasing a contamination risk. In this work, a single drop (8 μl) of heparinized whole blood could be efficiently filtered using a capillary effect without any external driving forces and without dilution. The blood storage in heparin tubes during 24 h at 4 °C initiated the formation of small crystals that formed auto-filtration structures in the sample upon entering the 3D-printed device, with pores smaller than the red blood cells, separating plasma from the cellular content. The total filtration process took less than 10 s. The presented printed plasma filtration microfluidics fabricated with a rapid prototyping approach is a miniaturized, fast and easy-to-operate device that can be integrated into healthcare/portable systems for point-of-care diagnostics.

Pharmacological Screening of Trachyspermum ammi for Antihyperlipidemic Activity in Triton X-100 Induced Hyperlipidemia Rat Model.

Background:Mortality rate is increasing due to cardiovascular problems throughout the world. These cardiac problems are directly associated with dyslipidemia.Aim:The aim of this study was to evaluate the antihyperlipidemic effect of aqueous extract and methanol extract of Trachyspermum ammi at 1 g/kg, 3 g/kg, and 5 g/kg dose levels in rats.Materials and Methods:For this purpose, 45 male albino rats were used and randomly divided into nine equal groups (n = 5). The lipid levels were increased after 24 h of single intraperitoneal injection of Triton X-100 (100 mg/kg) in rats. Aqueous and methanol extracts equivalent to 1 g/kg, 3 g/kg, and 5 g/kg were administered orally to the rats for 21 days. Atorvastatin (10 mg/kg) was used as standard drug. Blood samples were collected at 0, 2nd, 9th, 16th, and 23rd day by a direct cardiac puncture in Vacuette® heparin tubes. Serum was separated and then analyzed for lipid profile, liver function test (LFT), and renal function test (RFT) using standard diagnostic kits.Results:Results showed that extracts at 3 g/kg and 5 g/kg decreased the levels of total cholesterol, triglyceride, and low-density lipoprotein and increased high-density lipoprotein concentration in serum. T. ammi also decreased LFT and RFT parameters at the end of the study.Conclusion:T. ammi possessed antioxidant and antihyperlipidemic activities along with hepato- and nephro-protective effects.SUMMARYAqueous and methanol extracts of T. ammi were administered orally at 1-, 3-, and 5 g/kg doses to hyperlipidemic rats (Triton X-100 induced hyperlipidemia) and atorvastatin (10 mg/kg, orally) was used as standard drug. Methanol extract at 5 g/kg showed antihyperlipidemic effect that is identical to that of standard drug.Abbreviations Used: LDL: Low-density lipoprotein; TC: Total cholesterol; VLDL: Very low-density lipoprotein; HDL: High-density lipoprotein; T. ammi: Trachyspermum ammi; WHO: World Health Organization; CAD: Coronary artery disease; BHT: Butylated hydroxytoluene; BUN: Blood urea nitrogen; AST: Aspartate transaminase; ALT: Alanine transaminase; IP: Intraperitoneal.

Curious Case of Profound Thrombocytopenia in a Pregnant Woman

Introduction:Pseudo-thrombocytopenia (PTCP) is a benign artifact which generates anxiety in the patients and physicians resulting in unnecessary investigations. We report a case with profound artefactual thrombocytopenia.Case:A 31-years-old female in 38th week of gestation was referred to hematology clinic for evaluation of a platelet count of 48x109/L. A month ago platelet count was 102x109/L. She denied any symptoms. Examination was normal with no ecchymosis or petechiae. Laboratory work showed a white cell count of 11.2x109/L, hemoglobin 10.3g/L, platelet count 8x109/L and no hemolysis. Peripheral blood smear showed numerous platelet clumps (Figure 1). Estimated manual platelet count was 200x109/L. To our surprise clumps and low machine count were seen in samples drawn in sodium citrate and heparin tubes as well.Discussion:Ethylenediaminetetra-acetic acid (EDTA) dependent PTCP is a rare phenomenon with incidence of 0.09%-0.21% in general population (1). It is present in healthy subjects (2), severely ill patients with sepsis (3), autoimmune, neoplastic and liver diseases (4). PTCP may persist for 15-20 years without any clinical manifestations (5). Platelet aggregation in PTCP is due to anti-platelet antibody mediated in-vitro activation via GPIIb receptors (6). Antibodies may be IgG, IgA or IgM (5). It may be seen in samples drawn in citrate (5) heparin (7) and sodium-oxalate (8). One should suspect PTCP when there is fall in platelet count (usually <100x109/L), time-dependent spurious elevation of white cells(9), a normal mean platelet volume (4) or microscopic detection of platelet aggregates (1) in a patient without clinical manifestations. There is a rare report that addition of amikacin could inhibit and dissociate pseudo platelet aggregation in multianticoagulant-dependent pseudo-thrombocytopenia and EDTA-induced pseudo-thrombocytopenia (10). Early identifications is extremely essential when therapeutic decision making hinges on platelet count viz. management of acute myocardial infarction (11).Use of samples at 37°C (2), ammonium oxalate (5, 7), addition of amikacin and peripheral smear review may help in complicated cases.

Effect of various anticoagulants on the bioanalysis of drugs in rat blood: implication for pharmacokinetic studies of anticancer drugs.

BackgroundPharmacokinetic studies are vital in development and optimization of drugs. While blood samples can be collected either in EDTA, heparin or citrate containing tubes for the estimation of drug levels in plasma, EDTA tubes are more commonly used. The purpose of this study was to evaluate the effects of anticoagulants on bioanalysis of drugs. Six drugs used extensively in cancer therapy were selected. Albino wistar rats (N = 6 per drug) were dosed with one of the following drugs intraperitoneally—pemetrexed (50 mg/kg), imatinib (50 mg/kg), erlotinib (25 mg/kg), meropenem (60 mg/kg), 6-mercaptopurine (20 mg/kg) and voriconazole (6 mg/kg). Blood samples were collected 2 h after dosing (1 h in 6-mercaptopurine group due to short half-life) by terminal bleeding from the retro-orbital plexus. Blood was collected in each of Disodium ETDA, heparin, trisodium citrate (TSC) and no anticoagulant (plain) tubes. Drug levels in these samples were determined by validated HPLC assays. ANOVA with Tukey’s post hoc test was performed to identify statistically significant differences in drug concentrations in anticoagulant tubes. p < 0.05 was considered statistically significant.ResultsSignificant differences in concentration between anticoagulant tubes was observed in case of erlotinib (p = 0.013) and meropenem (p = 0.00), while borderline statistical significance for pemetrexed (p = 0.076). TSC tubes overestimated erlotinib levels, heparin tubes underestimated meropenem concentrations and EDTA tubes overestimated pemetrexed concentrations.ConclusionsCareful selection of anti-coagulant is necessary for accurate characterization of pharmacokinetics of drugs. Routine use of EDTA tubes may lead to erroneous interpretation of pharmacokinetic data.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3770-4) contains supplementary material, which is available to authorized users.

The preanalytical stability of glucagon as measured by liquid chromatography tandem mass spectrometry and two commercially available immunoassays.

Background One of the main challenges in the measurement of glucagon is the premise that it is unstable in human plasma. Traditionally, protease inhibitors have been used to prevent its degradation; however, their use is controversial. Here, we investigated the optimal method of sample collection for glucagon, with measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) and two commercially available immunoassays. Methods Blood from healthy fasting volunteers (n = 10) was processed under a variety of preanalytical conditions including collection in EDTA vs. lithium heparin tubes and the addition of aprotinin and/or a dipeptidyl-peptidase IV (DPPIV) inhibitor. Additionally, the effect of freeze thaw was assessed. Plasma glucagon concentrations were measured by LC-MS/MS and two commercially available immunoassays (HTRF® sandwich immunoassay, Cisbio and Milliplex MAP Human Metabolic Hormone Panel, Merck Millipore). Results A systematic bias of Milliplex > LC-MS/MS > HTRF was noted and plasma glucagon concentrations were significantly different between methods (Milliplex vs. LC-MS/MS P < 0.01; Milliplex vs. HTRF P < 0.0001; LC-MS/MS vs. HTRF P < 0.001). The addition of aprotinin, DPPIV inhibitor or a combination of aprotinin and DPPIV inhibitor had no effect on plasma glucagon concentrations when compared to 'non-stabilized' samples or each other. Whether samples were taken in EDTA tubes or lithium heparin tubes made no difference to plasma glucagon concentrations. These findings were consistent for all three methods. Plasma glucagon concentrations were not significantly different after two freeze-thaw cycles (performed on samples in EDTA tubes containing aprotinin and DPPIV inhibitor). Conclusions This study demonstrates that glucagon is stable in both EDTA and lithium heparin tubes when stored at -80℃. Furthermore, the addition of aprotinin and DPPIV inhibitors is unnecessary.

INTERLEUKIN-10 PLASMA DAN LIMFOSIT-T CD4+ PENDERITA TERINFEKSI HIV

HIV &amp; AIDS have become a global problem throughout the world including Indonesia. The incidence is rapidly increasing. Various treatment and efforts have been carried out but until now have not yet been succeeded, the mortality remains high. Examination of CD4+ lymphocyte-T which is carried out to determine the immune status and monitoring the treatment has some limitation. Besides the degradation of CD4+ lymphocyte-T, the increase of the disease progression is also followed by an increase of interleukin-10 as well. The determination of interleukin-10 is expected to be use as an alternative examination if CD4+ lymphocyte-T can not be performed. To analyze the correlation between the levels of plasma interleukin-10 and amount of CD4+ lymphocyte-T in the asymptomatic HIV infected patients. A cross sectional, observational analytical study of 41 patients with HIV infection stage I, has been conducted from March–April 2009, at the Intermediate Infectious Disease Care Unit Dr. Soetomo General Hospital, Surabaya. The diagnosis of HIV patients was based on positive result of HIV test using three (3) different methods. Eight mL venous blood were taken from each patient, 6 mL is put into a heparin tube for the examination of IL-10 plasma with ELISA method and 2 mL is put into a K3EDTA tube for examining the CD4+ lymphocyte-T using flowcytometry. The results showed of plasma IL-10 level and the amount of CD4+ lymphocyte-T, which were analyzed by Pearson correlation test to determine the correlation between the two (2) variabels. The IL-10 levels in HIV patients were 3.80–44.50 pg/mL (mean 18.09 pg/mL, SD 8.84 pg/mL). The amount of absolute CD4+ lymphocyte-T was 5–846 cells/µL (mean 322.07 cells/µL and SD 221.89 cells/µL), while the amount of percentage CD4+ lymphocyte-T was 0.41–29.48% (mean 13.99%, SD 7.62%). Statistical analyzes show a significant negative correlation either between plasma IL-10 level with absolute CD4+lymphocyte-T and plasma IL-10 level with percentage of CD4+ lymphocyte-T, e ach level of r was –0.652 and –0,683. A significant negative correlation was also formed between plasma IL-10 level and the amount of CD4+ lymphocyte-T in the HIV infected patients. In the HIV infected patients, the increase of plasma IL-10 level was followed by a decrease of CD4+ lymphocyte-T.