Heparin Tubes Research Articles

Effects of different tube types on patient classification using current diabetes decision limits

Management of diabetes is a challenge starting in the pre-analytical phase with selecting the most appropriate glycolysis inhibitor. Study goal was to calculate the impact of tubes with different glycolysis inhibitors on the classification of the glycemic control of 157,415 consecutive hospital patients according to current WHO diabetes criteria. MethodsGlucose and lactate were measured in parallel in samples from 68 healthy subjects collected and stored in different sample tubes from Sarstedt and Greiner. Bias to baseline conditions (fluoride heparin (FH) tubes, centrifugation within 1 h) was determined. ResultsIn baseline samples, glucose concentration in fluoride/EDTA/citrate (FC) plasma was ~13% higher and lactate concentration ~20% lower compared to FH, fluoride oxalate, and fluoride EDTA plasma, and in serum. Glucose recovery after storage up to 48 h was 99–101% in the different tubes, but the effectiveness of glycolysis inhibition by FC was inconsistent. Based on the observed mean bias of 12% when FC tubes are used, we estimate an increase of 48.4–55.8% in the frequency of patients with impaired glucose levels using current WHO criteria. ConclusionUsing current established decision limits, the number of patients with impaired glucose levels in the hospital would increase substantially with a strong impact on patient treatment and consumption of resources. The unpredictable failure of glycolysis inhibition in FC tubes does not allow to adjust the decision limits by a fixed factor. In the absence of prospective outcome studies with FC tubes, we recommend to measure glucose in samples containing FH.

Reversible platelet aggregation in the presence of calcium ions: mechanisms and potential value

Disorders in the functions of platelets – blood cells responsible for the blood clots formation and prevention – are observed as independent diseases, as a complication of cancer and hematological diseases or as a result of a therapy. Nowadays, a test of platelet aggregation by aggregometry is the only diagnostic method for assessing the platelets functions. There are several varieties of aggregometry, which differ both in the method of recording the formation of platelet aggregates and in the method of preparing platelets for the experiment. In most laboratories, it is customary to conduct aggregometry in platelet-rich plasma in the presence of citrate ions. In this case, the concentration of calcium ions in plasma decreases, it prevents the thrombin formation and the plasma coagulation. On the other hand, it has long been known that platelet aggregation in response to ADP in the presence of calcium ions (in blood plasma collected in heparin or hirudin tubes, also blocking plasma clotting) is reversible: after 1-5 minutes after the addition of the activator, the disaggregation begins until the light transmission of the solution (platelet concentration) returns to its original level. This phenomenon is called "reversible” platelet aggregation. Reversible aggregation (“disaggregation”) is sometimes observed in aggregometry of citrate plasma, especially in pediatric patients. However, it is usually not considered normal and is considered a sign of platelet dysfunction. This review considers the known mechanisms of disaggregation in the presence or absence of calcium ions in the medium. The role of secondary activation of platelets as a potential cause of irreversible aggregation is discussed, as well as possible versions for explaining the results of aggregometry, when reversible platelet aggregation is observed.

Abbott ARCHITECT STAT Contemporary Troponin I Outlier Detection and Prevention of Erroneous Outlier Result Reporting Using an Immediate Repeat Testing Protocol: A 1-Year Summary

Abstract Objectives The laboratory sought to monitor and eliminate reported patient cardiac troponin outlier results using the Abbott ARCHITECT STAT Contemporary Troponin-I test. Methods The laboratory implemented an immediate repeat test for all patient samples yielding an Abbott ARCHITECT STAT contemporary Troponin I concentration greater than the combined male and female 99th percentile of 28 ng/L. The lower troponin concentration of the paired testing was reported. Samples with reference range troponin concentration were reported without repeat testing. Blood collected in Becton Dickinson Vacutainer PST Gel and Lithium Heparin tubes was centrifuged using a HemoCue StatSpin Express 4 centrifuge 4,000 g × 5 minutes and immediately tested on the primary tube. Troponin outliers were defined as paired samples showing a percentage change based on the low value of the pair exceeding the significant change threshold of 4.7x%CVanalytical for brackets of troponin concentrations (P0.0005), representing a significant change value for serial tests of 1.41xZxCVa%, where Z is 3.3. Critical troponin outliers were sample pairs with one result exceeding and one result within the 99th percentile reference range. Turnaround time was defined as time of receipt to time of verification. Results The outlier rate, based on 4,575 troponin tests performed on 3,797 patient samples, was 0.48% (N = 22) of troponin determinations, 0.58% of test samples, and critical outliers 0.13% (N = 6) of troponin determinations, 0.16% of test samples. The significant change threshold at 28 ng/L was 37%. The worst outlier pair was 5,978 ng/L, true value 3,184 ng/L; critical outlier pair 146 ng/L, true value of 14 ng/L. Twenty-one percent of test samples were tested in duplicate; 777 samples had troponin concentration >28 ng/L. Median and mean turnaround time were 32 minutes; 90% of orders completed in 54 minutes. Conclusion The laboratory detected and prevented reporting erroneously high outlier troponin results in 0.58% and critical outlier troponin results in 0.16% of test samples using an immediate repeat testing protocol.

Clinically feasible method for assessing leukocyte rheology in whole blood

This study reports a novel method for assessment of leukocyte rheological activation with a new designed microchannel array chip to mimic the human microvascular network for microchannel array flow analysis (MCFAN). Study subjects were 79 healthy volunteers and 42 patients with type 2 diabetes mellitus (DM) and 36 patients with acute coronary syndrome (ACS). Using the anticoagulants heparin and ethylene-diamine-tetraacetic acid (EDTA)-2Na which inhibits platelets and leukocytes by chelating Ca2+, we were able to quantify leukocyte rheological activation by the subtraction of passage time of blood treated with both heparin and EDTA-2Na from that of blood treated with heparin only. We confirmed that passage times of whole blood with heparin + EDTA-2Na were always shorter than those of whole blood with only heparin in healthy subjects and patients with DM or ACS under suction pressures of − 30 cmH2O. There was a significant correlation between delta whole blood passage time {(heparin tube) − (EDTA-2Na + heparin)} and serum levels of myeloperoxidase and adhesive leukocyte number, respectively, even in blood from patients with DM or ACS, who suffered from inflammation. In conclusion we have developed a clinically feasible method for assessing leukocyte rheological activation in whole blood in ex vivo.

Lithium heparin tubes should not be used for the determination of gamma-glutamyl transferase activity

Lithium heparin tubes should not be used for the determination of gamma-glutamyl transferase activity

Stabilization of Plasma Glucose: The Use of Newer Technology and Pragmatic Laboratory Practice.

The target values for plasma glucose concentrations for the investigation and diagnosis of diabetes and impaired fasting glucose, and the realization that small incremental changes in glucose concentration increase the risk of adverse events, has led to greater focus on laboratory glucose results. Although analytical methods show acceptable precision, the control of preanalytical error due to the stability of glucose remains problematic. The aim of this study was to compare glucose concentrations in 3 different and commercially available blood tubes, with analysis and storage under current practices and conditions. Blood samples for glucose were obtained from consenting patients attending the Diabetic Clinic at the Royal Victoria Hospital, Belfast. Blood was collected into BD Vacutainer® Barricor™ Lithium Heparin tubes, BD Vacutainer Fluoride EDTA tubes, and Greiner Vacuette® FC-Mix (sodium fluoride/citrate/Na2EDTA) tubes in that order. The Barricor tubes were immediately centrifuged at 4000g for 3 min. All samples were then sent to the Biochemistry Laboratory for analysis on the same day, and again the following day after storage at 4 °C. There was no significant difference in mean glucose concentrations between immediately centrifuged Barricor and FC-Mix tubes when analyzed on day 0. Both tube types demonstrated higher mean glucose concentrations than traditional fluoride EDTA (F/EDTA) samples. Both immediately separated Barricor and citrated FC-Mix plasma preserve glucose concentrations to the same extent, and better than F/EDTA preservative. These newer technologies involved offer pragmatic solutions to improved glucose analysis, allowing laboratories to choose the best option given the source of their samples.

Assessment of the Stability of Supraphysiological Ascorbate in Human Blood: Appropriate Handling of Samples from Clinical Trials for Measurements of Pharmacological Ascorbate.

Based on encouraging results from several early-phase clinical trials, there is renewed interest in the use of pharmacological ascorbate (i.e., intravenous administration resulting in >≈10 mM plasma ascorbate concentrations) in combination with standard-of-care cancer treatments including radiation and/or chemotherapy. Under normal, healthy physiological conditions, humans maintain plasma ascorbate concentrations in the range of 40-80 lM. However, in vivo antitumor activity requires supraphysiological plasma concentrations on the order of ≈20 mM. The stability of ascorbate in whole blood has been well studied. The goal of this work was to determine the appropriate handling methods of blood samples, after treatment with pharmacological ascorbate, which allow for the optimal measurement of ascorbate in plasma for dosing verification. Our findings indicate that ascorbate concentrations (mM) are relatively stable in whole blood collected in sodium heparin tubes and stored on ice (or at 4°C) for up to 24 h. After 24 h, ascorbate levels in plasma are relatively stable at 4°C for up to 72 h. At -20°C, plasma concentrations are relatively stable for 2-3 weeks, while at -80°C, ascorbate concentrations in plasma are stable for at least one month. In contrast, patient samples showed better stability when stored as whole blood compared to plasma at 4°C but increasing hemolysis over time may significantly skew ascorbate measurements. Additionally, patient samples can be reliably stored as plasma at -20°C for up to three weeks in either a frost-containing or frost-free environment. This information can guide the collection, processing and storage of clinical samples after pharmacological ascorbate infusions amenable to multi-center clinical trials.

Myeloid-Derived Suppressor Cells in Lung Transplantation.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immune cells from the myeloid lineage. MDSCs expand in pathological situations, such as chronic infection, cancer, autoimmunity, and allograft rejection. As chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation (LTx), MDSCs may play a role in its pathophysiology. We assessed phenotype and frequency of MDSCs in peripheral blood from lung transplant recipients and its relationship to post-transplant complications and immunosuppression. Granulocytic (G)-MDSC were identified and quantified by flow cytometry of blood from 4 control subjects and 20 lung transplant patients (stable n = 6, infection n = 5; CLAD n = 9). G-MDSC functionality was assessed in vitro by their capability to block CD4 and CD8 T cell proliferation. More G-MDSC could be assessed using EDTA tubes compared to heparin tubes (p = 0.004). G-MDSC were increased in stable lung transplant recipients vs. non-transplant controls (52.1% vs. 9.4%; p = 0.0095). The infection or CLAD groups had lower G-MDSCs vs. stable recipients (28.2%p = 0.041 and 33.0%; p = 0.088, respectively), but were not different among CLAD phenotypes. G-MDSC tended to correlate with cyclosporine A and tacrolimus levels (r2 = 0.18; r2 = 0.17). CD4 and CD8 cells proliferation decreased by 50 and 80% if co-cultured with MDSCs (1:6 and 1:2 MDSC:T-cell ratio, respectively). In conclusion, circulating MDSCs are measurable, functional and have a G-MDSC phenotype in lung transplant patients. Their frequency is increased in stable patients, decreased during post-transplant complications, and related to level of immunosuppression. This study may pave the way for further investigations of MDSC in the context of lung transplantation.

Haemolysis and lipemia interfere with resistin and myeloperoxidase BioVendor ELISA assays

IntroductionThe aim of our study was to investigate the influence of haemolysis and lipemia on resistin (RES) and myeloperoxidase (MPO) measurement by BioVendor enzyme-linked immunosorbent assays (ELISA).Materials and methodsBlood was taken from healthy volunteers into lithium heparin tubes. Plasma samples were spiked with Lipofundin® emulsion (B. Braun Melsungen AG, Germany) for lipemia interference testing. Haemolysed samples were obtained by drawing aliquots of heparinized blood through a 26 gauge needle. Index of haemolysis (H), lipemia (L) and triglyceride concentration were measured on Abbott Architect c8000. Haemoglobin concentration was measured on Sysmex XN-1000. Concentrations of RES and MPO in all samples were determined with RES and MPO ELISA kits (BioVendor, Czech Republic). All measurements were performed in triplicate. Biases from the native samples were calculated for both analytes and compared with an arbitrary value (e.g. ± 10%).ResultsTriglyceride concentration in the investigated samples ranged from 0.57 to 38.23 mmol/L, which corresponds to L index from - 0.01 to 13.77. Haemoglobin concentration in all samples ranged from 0 to 8 g/L which correspond to H index from 0.05 to 8.77. Both MPO and RES showed significant biases at 1 g/L haemoglobin (58.7% and 66.7%, respectively). Also, both MPO and RES showed significant biases at 4.66 mmol/L triglycerides (33.8% and - 12.2%, respectively).ConclusionsResistin BioVendor assays are affected by haemolysis and lipemia already at low degree of interferent. Haemolysis was found to interfere at 1 g/L haemoglobin for both assays, while lipemia interferes at 4.66 mmol/L of triglycerides.

A Human Model of Whole‐Body Ischemia‐Reperfusion: Changes in Exosome Release

In‐depth knowledge of individual patients' physiology impacts perioperative management, which also impacts efficacy and safety of surgical procedures. Exosomes are a new paradigm in cellular communication, cell stress, and biomarker development that have only recently begun to be addressed in the surgical population. Exosomes, secreted by almost all cells, carry biologically active molecules including DNA, RNA, proteins, lipids, cytokines, growth factors and metabolites. The role of exosomes in the surgical population is not well known and may have major impact on perioperative care and monitoring.Deep hypothermic circulatory arrest (DHCA) is a profound perioperative stress event involving isolation of the systemic vasculature, arrest of the circulation to major organ systems, and whole body ischemia‐reperfusion. DHCA is used for surgical repair of the great vessels including pulmonary thromboendarterectomy (PTE) for which the University of California, San Diego (UCSD) serves as a leading center. Our study tests the novel hypothesis that surgically induced DHCA increases the release of exosomes, alters exosome content and changes the ability of exosomes to affect cellular metabolism and function.Arterial blood was obtained at 5 timepoints in patients undergoing DHCA for PTE: immediately after induction of anesthesia (baseline), after initiation of cardiopulmonary bypass (CPB), at completion of cooling to body temperature of 20C, after circulatory arrests, and after patient is rewarmed and off CPB (chest closure). Blood was centrifuged in heparin tubes to prepare plasma for exosome isolation and nanoparticle tracking analysis (NTA). Additional exosome samples were stored for further immunoblotting, electron microscopic, proteomic, microRNA and in vitro analysis.Particle concentration as measured by NTA immediately after initiation of CPB and at the completion of cooling was decreased from baseline. Thereafter, particle concentration showed an increase after DHCA and a further increase during chest closure at the conclusion of the surgery. Mean particle sizes at each timepoint were within the known size distribution of exosomes.In conclusion, our data show that initiation of CPB and cooling can decrease exosome levels in blood, while whole body ischemia‐reperfusion associated with DHCA in patients is a stimulus for exosome release. Future work will focus on the changes in the proteome and microRNA content of exosomes after DHCA and the physiological consequences of exosome release on intact in vitro cell models. This model also lends itself well to further detailed investigation of tissue and organ specific exosomal responses to ischemia‐reperfusion.Support or Funding InformationR21 AG058174‐01A1 (PI: D Roth) 09/01/2018 – 06/30/2020 Funding Source: NIH/NIA T32 GM121318 (PI: HH Patel; co‐PI: DM Roth) 07/01/16‐06/30/22 Finding Source: NIH/NIGMSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blood monocyte profiles in COPD patients with PiMM and PiZZ α1-antitrypsin.

Human blood monocytes are divided into populations based on the differential expression of CD14 and CD16 receptors: CD14 + CD16(classical), CD14 + CD16 + (intermediate), and CD14-CD16+ (non-classical). Given their functional differences and their role in pathogenesis of chronic obstructive pulmonary disease (COPD), monocyte profiling is of clinical interest. Here we investigated blood monocyte subsets in clinically stable COPD patients with alpha1-antitrypsin (AAT) deficiency (PiZZ, n=7) and with normal AAT variant (PiMM, n=7). Peripheral whole blood was collected in sodium heparin tubes and incubated with LPS (from E. coli; 1μg/ml) or placebo for 6hat 37°C, 5% CO2. To profile monocyte subsets we performed flow cytometry analysis based on HLA-DR and CD14/CD16 staining. HLA-DR+subsets of cells did not differ between PiZZ and PiMM COPD, and healthy controls (n=7), used as a reference. Monocyte profiling, which express the CD14 and CD16, but not the HLA-DR (HLA-DR-) showed that intermediate monocytes subset was lowest in PiZZ group, and almost totally disappeared from blood treated with LPS. The non-classical subset was almost absent in PiZZ patients independently of LPS treatment. Recent studies demonstrate that non-classical monocytes exhibit a unique ability to protect the vascular endothelium under both homeostatic and inflammatory conditions whereas intermediate monocytes are recruited at a later stage of inflammation, and are associated with secretion of cytokines/chemokines and wound healing. Evident alterations in blood monocyte subsets together with a partial reduction of AAT levels, an important anti-inflammatory protein, can be key factors for the early manifestation of emphysema in some PiZZ AATD carriers.

Evaluation of BD Barricor™ Plasma Blood Collection Tube for Biochemical Tests.

Blood from patients who are receiving anticoagulant therapy may take longer to clot. Anticoagulation is an important component of the dialysis prescription. We compared BD Vacutainer® Barricor™ Plasma Blood Collection Tubes (BD BarrricorTM), BD Vacutainer® PST™ Lithium Heparin Tubes (BD PST™), and BD (Becton-Dickinson, Franklin Lakes, NJ, USA) Vacutainer Serum Separator Tubes (BD SST), as reference tube in dialysis patients to examine whether they had an effect on routine biochemical tests. A total of 29 chronic hemodialysis patients were included in this study. Samples were collected into BD BarrricorTM, BD PST™, and BD SST tubes after the dialysis. All the tubes were centrifuged by NF 1200R rotor (1,300 g for 10 minutes at 22°C, 1,200 g for 10 minutes at 4°C, 2,400 g for 10 minutes at 22°C, respectively) after the incubation period. Eleven routine clinical chemistry parameters (Creatinine, Urea, Na, K, Cl, AST, ALT, Total Bilirubin, Direct Bilirubin, Calcium, Cholesterol) were analyzed on a Beckman Coulter AU 5800. Results of creatinine, K and cholesterol were statistically significantly different between the SST and LiH (p = 0.014, p = 0.009, and p < 0.001, respectively). In terms of other biochemical parameters we tested for all three tubes there was no clinically significance inspite of the statistically significance. BD Barricor™ tubes provide fast, clean, high-quality plasma samples, safe results, and may lower times and costs.

Review and Performance Comparison of Lead-Free Shields and Lead Shields, In Terms of Biological Effects in Nuclear Medicine by the Comet Method

Introduction: Ionizing radiation can cause damage to biologically important DNA molecules. Radiation in nuclear medicine radiation exposes people working in from two main positions; administer in gradiopharmaceuticals (the barn and squeezing a generator, procurement, distribution and drug infusion) and patients that receive radiation treatment (patients that are prescribed a radiopharmaceutical). Use of lead aprons is disputed practice in radiation treatment due to the creation of special lead X-rays of high-energy gamma ray collisions and the high absorption coefficient of such radiations in the body. Correct use of personal protective equipment, such as uniforms and flexible protection, and compliance with radiation protection can greatly reduce damage. Flexible lead shields are usually made from materials such as rubber; polymers or elastomers (polymers that are elastic) are used as the basis for fine lead particles. The problems with lead shields have led to recent discussion of the use of lead-free shields. If the biological effects caused by lead-free shields give less than two states with and without a lead shield, the shield that is lighter, weaker and that doesn’t cause environmental pollution, can be used in place of lead. Materials and Methods: To review and achieve the above objective, blood samples were taken from non-radiation operatives and poured in heparin tubes, and then placed in the considered distance of time and place; after radiation, they were exposed to gamma radiation resulting from technetium, comet assay will be performed on them. Results: density was super less dense compared to the lead and was about one-tenth of the density of lead (1.23). DNA damage in both groups (with and without a lead shield) is greater when compared with the control group. Conclusion: By using two-way analysis of variance, the time variable was significantly effective in increasing the damage. In addition, the type of shield variable (lead or lead-free) and meaningful distance have not shown.

Unexpected Low Expression of Platelet Fibrinogen Receptor in Patients with Chronic Myeloproliferative Neoplasms: How Does It Change with Aspirin?

INTRODUCTIONPatients affected by Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered at high risk of thrombo-haemorrhagic events, but the role of the platelet count in the assessment of the risk of vascular events is still controversial. A tight correlation was found between the platelet count and plasma sCD40L, which appears to be required for thrombus formation in vivo. However sCD40L is increased both in MPNs and reactive thrombocytosis. Intravascular aggregates of platelets and leukocytes, mediated by P-selectin and CD11b on the former and the latter, respectively, have been observed.Both of this processes should imply a perpetual - and measurable - platelet activation. Several studies on platelet function have been already proposed, nevertheless the mechanisms through which platelets are able to trigger vascular events, are not yet adequately clarified.A refined method for the determination of platelet activation appears to be the use of platelet PAC-1 antibody, able to identify the expression of the fibrinogen receptor of platelet glycoprotein IIb/IIIa. This expression is indeed unique in the process of platelet activation, and yet rarely analyzed. Moreover, since the platelet fibrinogen receptor exposure seems to be influenced by turbulence in blood flow, we have thought that its evaluation could provide important biological evidences to explain some clinical manifestations, such as microvascular disturbances.METHODSBlood samples from 40 consecutive MPNs patients who never received cytoreductive agents, were obtained. 28/40 patients were receiving a continuative antiplatelet prophylaxis with low dose aspirin (ASA, 75-100 mg) at the time of collection, while 12/40 of them were not on such therapy. Our aim was to verify the expression of platelet fibrinogen receptors (PFRs) in the two different groups of patients compared to healthy volunteers, using whole blood flow cytometry. In each experiment sodium citrate and heparin (positive control of platelet activation) tubes were collected from the same patient. Within 10 minutes from blood sampling, 5 ml of whole blood from each tube was incubated for 20 minutes at room temperature in the dark with saturating concentration of CD61 PerCP, CD62P PE and PAC-1 FITC. Positive control was also incubated with PAC-1 in the presence of Arg-Gly-Asp-Ser (RGDS) in order to test the specific antibody binding. Samples were fixed with paraformaldehyde 1% for 30 minutes at 4°C in the dark and analyzed on a flow cytometer.RESULTSSurprisingly, we have been able to verify a very low PAC-1 binding to platelets in patients with MPNs not receiving cytoreduction nor antiplatelet agents (33%) if compared to that observed in healthy subjects (61%; p<0.0001). The use of aspirin seems conversely to restore the expression of platelet fibrinogen receptor, as PAC-1 binding capacity is comparable to that of healthy volunteers (56%). No difference was found with respect to the JAK2 Val617Phe mutation and its allele burden. Interestingly, by focusing on the group of patients under antiplatelet prophylaxis and with no history of thrombosis, it was found that subjects with persistent microcirculatory disorders show a higher PAC-1 binding capacity if compared to the asymptomatic ones (67% vs 52%, p= 0.04).CONCLUSIONIn untreated MPNs, a large amount of platelets are resting in a conformation that is unable to bind fibrinogen, as demonstrated by the low PAC-1 expression in cytofluorimetry. This lack of activity can be reversed by administering ASA, which is also known for its fibrinolytic and hypoprothrombinemic effects. We hypotesize that the hypercoagulable states observed in these patient could depend on a primarly plasma-driven impairment of fibrin turnover and thrombin generation. Further investigations are going to be promoted by our Group. PAC-1 could be at the same time a good marker of aspirin resistance in patients experiencing microcirculatory disorders. [Display omitted] DisclosuresMartinelli:Janssen: Consultancy; Jazz Pharmaceuticals: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Novartis: Speakers Bureau.

Performance and variability of QuantiFERON Gold Plus assay associated with phlebotomy type.

BackgroundQuantiFERON Gold Plus (Plus) assay has two approved methods for blood collection: direct in-tube (Plus direct) or the transfer of blood from a lithium heparin tube (Plus transfer). Currently, there is little data comparing the results of Plus and the QuantiFERON Gold In-Tube (Gold) based on blood collection.MethodsIn 2017, high risk healthcare workers undergoing annual tuberculosis infection screening at Houston Methodist Hospital, a private hospital in the Texas Medical Center (Houston, TX, U.S.A.) were consented and enrolled in a study comparing the Gold-in-tube (Gold), Plus direct in-tube, and Plus transfer assays. Blood was drawn concurrently for all 3 assays.ResultsPhlebotomy occurred on 300 consecutive, consented and enrolled participants in the study. The proportion of positive test results for the Gold, Plus direct and Plus transfer assays were 10% (29/300), 12% (35/299) and 17% (51/299), respectively. The agreement in the results of Gold versus Plus direct, Gold versus Plus transfer, and Plus direct versus Plus transfer was 91%, kappa (κ) = 0.56; 91%, κ = 0.59; and 85%, κ = 0.37, respectively.ConclusionsAmong high risk healthcare workers in a low prevalence tuberculosis setting, the Gold Plus assay had a higher proportion of positive results than the Gold in-tube assay. The agreement between the Gold, Plus direct and Plus transfer assays was unexpectedly low for simultaneously obtained samples. Blood transfer using lithium heparin offers individual clinics and public health programs greater ability to customize protocols, but variability of results still exists.

Bilirubin Interference in Plasma Amino Acid Analysis by Ion Exchange Chromatography.

To evaluate the effect of bilirubin interference on plasma amino acid analysis by Ion Exchange Chromatography (IEC). Cross-sectional (method validation) study. Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi from August 2016 to July 2017. Twenty non-icteric samples from paediatric patients were collected in lithium heparin tubes and analysed for amino acids on IEC-based Biochrome® 30+ Analyzer (Harvard Biosciences UK). Baseline bilirubin levels were noted. Samples were spiked with neonatal bilirubin standard with concentration of 488.4 mol/L (Spinreact®-Spain) at final concentrations of 50, 150 and 230 mol/L and re-analysed for amino acids at these three concentrations. Among the 20 selected patients with normal amino acid profiles, 12 (60%) were males. Majority (55%) were in age group of 1-5 years. Significant difference was observed for Arginine (p = 0.01), Histidine (p = 0.001), Isoleucine (p = 0.01), Leucine (p = 0.007), Lysine (p = 0.005), Ornithine (p = 0.03) and Phenylalanine (p = 0.02). Mean rank of these amino acids showed decreasing trend with the increase of bilirubin concentration, and pronounced interference was identified at bilirubin level of 50 mol/L. No difference was observed for alanine, citrulline, glutamic acid, glycine, methionine, proline, threonine, tyrosine, asparagine, aspartic acid, cystine, valine and tryptophan. Bilirubin significantly interferes with certain amino acid levels when analysis is carried out by ion exchange chromatography. A close follow-up of such patients with other biochemical tests and a repeat amino acid analysis, after jaundice is settled, is recommended to confidently rule out any possible inherited metabolic disorder in these patients.

Influence of sample collection tube method, anticoagulant-containing plasma versus serum, on influenza virus hemagglutination inhibition titer and microneutralization titer serological assays

BackgroundThe hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development. It is unknown if the results can be influenced by sample type and anticoagulants. The purpose of this study was to evaluate the influence of different sample collection methods, in particular different anticoagulants, and choice of plasma or serum, on influenza virus serological assays.MethodsBlood samples from thirty donors previously immunized against influenza viruses were collected using six different types of blood collection tubes, two of which collect serum and four of which contain various anticoagulants for collecting plasma. Serum: (1) serum separator tubes (SST); and (2) Plus Plastic serum “red-top serum” tubes. Plasma: (3) spray-coated K2 ethylenediaminetetraacetic acid (EDTA) tubes: (4) Sodium Heparin tubes; (5) Citrate tubes with 3.2% sodium citrate solution; and (6) Glass Blood Collection tubes with acid citrate dextrose. Samples were tested against three different influenza viruses (A/California/07/2009 (H1N1pdm09), A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012) for hemagglutination inhibition titer and virus neutralization titer via a microneutralization (MN) assay, and data compared to that obtained for standard serum sample collected in SST.ResultsHAI and MN titers against type A viruses were within two dilutions compared to SST collection method over 96% of the time irrespective of sample type or anticoagulant. However, HAI titers for type B virus were more variable across different collection methods. EDTA plasma samples were greater than two dilutions higher than SST serum samples 70% (21 of 30 samples) of the time. In contrast, MN titers were within two dilutions over 96% of the time, with the highest deviation noted in acid citrate dextrose plasma samples (3 of 30 samples tested, 10%).ConclusionsThese data provide useful guidelines for sample collection and serology testing when screening: (i) influenza vaccine immunogenicity antibody response; (ii) antibody responses to newly emerging viral strains; and (iii) clinical samples for anti-influenza antibody activity.

Comparison Between K3EDTA and Lithium Heparin as Anticoagulant to Isolate Bovine Granulocytes From Blood.

Granulocytes play a key role in the defense against invading pathogens. To study granulocyte functions, the isolation of a pure and active cell population from fresh blood is required. Anticoagulants and red blood cells (RBCs) lysis used in the isolation procedure may influence cell harvest, cell marker expression, and pre-activation of cells. In this study, the influence of the anticoagulants K3EDTA or lithium heparin and the effect of different RBCs lysis methods on bovine granulocyte population from fresh blood of healthy cows after density gradient centrifugation were investigated. Venous blood from healthy cows was collected in K3EDTA and lithium heparin tubes. Density gradient centrifugation to separate granulocytes from other cells was conducted using Biocoll. Then, RBCs were lysed with hypotonic water or 0.2% sodium chloride (NaCl). Immediately after isolation, harvest, viability, size, granularity, purity, and CD11b expression as a marker for granulocytes was analyzed by flow cytometry. In addition, as a marker for activation and reactivity of the granulocytes, we stimulated cells with phorbol-myristate-acetate to evaluate the release of reactive oxygen species. Furthermore, extracellular trap (ET) formation was investigated by confocal immunofluorescence microscopy in untreated control cells and cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin. We did not find a significant difference in percentage of dead cells when comparing the two anticoagulants or the different RBCs lysis methods. However, the percentage of granulocytes in the harvested population was significantly less using lithium heparin blood as anticoagulant compared to K3EDTA. The granulocytes harvested from lithium heparin blood and water lysis exhibited higher clumping and pre-activation of unstimulated control cells as indicated by isolation of doublet cells, increased CD11b expression, and increased oxidative burst and higher amount of ET-releasing cells. Furthermore, the combination of K3EDTA as anticoagulant and NaCl as RBCs lysis method revealed the lowest variability and highest difference between untreated and methyl-β-cyclodextrin-treated cells when quantifying ET formation. In conclusion, density gradient centrifugation of K3EDTA blood resulted in higher purity of bovine granulocytes compared to lithium heparin blood. In contrast to water lysis, NaCl lysis method is recommended to avoid pre-activation of cells which may occur during hypotonic water lysis.

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.