Buffered Sodium Citrate Research Articles

In vitro analysis of n-acetylcysteine (NAC) interference with the international normalized ratio (INR)

Background Previous literature suggests a laboratory interference of n-acetylcysteine (NAC) with prothrombin time (PT) and the international normalized ratio (INR). Early publications focused on this interaction in the setting of an acetaminophen overdose and evaluated the INR of patients receiving intravenous NAC. However, there is limited literature describing the concentration-effect relationship of NAC to INR measurement in the absence of acetaminophen-induced hepatotoxicity at therapeutic NAC concentrations. The purpose of the study is to quantify the degree of interference of NAC on INR values at therapeutic concentrations correlating to each infusion of the regimen (ex. bag 1: 550 mcg/mL, bag 2: 200 mcg/mL, bag 3: 35 mcg/mL, double bag 3: 70 mcg/mL) and at supratherapeutic concentrations in vitro. Methods Blood samples were obtained from study volunteers. Each blood sample was transferred into vials containing 0.3 mL buffered sodium citrate 3.2% and spiked with various concentrations of NAC for final concentrations of 0, 35, 70, 200, 550, 1000, 2000, and 4000 mcg/mL. The samples were centrifuged and tested to determine PT and INR on two separate machines: Siemens CS-2500 and Stago SN1114559. We would require a sample size of 6 to achieve a power of 80% and a level of significance of 1.7% (two-sided). Differences between INRs at varying concentrations were determined by Friedman’s test. For multiple comparisons, post hoc analysis was performed using Wilcoxon signed-rank test with Bonferroni adjustment. Analyses were performed with SAS version 9.4 (SAS Institute, Cary, NC). Results Participants included 11 healthy subjects: 8 males, 3 females, median age 30 years (range 25 − 58). Median and interquartile ranges (IQR) INR for the baseline samples were 1.09 (IQR 1.05, 1.16) for Siemens and 1.03 (IQR 0.99, 1.11) for Stago analyzers. There was a significant difference in INR between the therapeutic concentrations (baseline, 35, 70,200, or 550 µg/mL) (Siemens p = .0008, Stago p < .0001). The 550 µg/mL concentration with the Siemens analyzer was the only one compared separately and found to be significantly greater than the baseline (1.07 vs 1.22, p = .02). For the Stago analyzer the 200 µg/mL and 500 µg/mL were compared and found to be significantly different from baseline (1.00 vs 1.07 and 1.19, adjusted p = .02 and p = .03, respectively). The largest INR increase seen was in one subject from a baseline of 1.07–1.32 with the 550 µg/mL concentration. Increases in concentrations to supratherapeutic levels resulted in a statistically significant non-linear increase in INR for all concentrations (Siemens p < .0001, Stago p < .0001). All of these concentrations were found to be significantly different from baseline (all adjusted p < .05). Conclusion Although it was found that at therapeutic concentrations the in vitro presence of NAC affects INR measurements on two different machines, the change is of little clinical relevance. Supratherapeutic concentrations of NAC affect INR significantly, but the clinical utility of those results is limited by the rarity of those concentrations being measured.

Circulating microparticles and activated platelets as novel prognostic biomarkers in COVID-19; relation to cancer.

Background and aimThe study aimed to determine whether the MPs levels and platelet activation are affected by the COVID-19 infection in both malignant and non-malignant patients compared to healthy individuals and define their contribution to the COVID-19 associated coagulopathy and the relation of these MPs to other hematologic parameters.Patients and methodsWe recruited 23 malignant patients with reverse transcription polymerase chain reaction (RT-PCR) positive COVID-19, also, 19 COVID-19 non-malignant patients, and 20 healthy volunteers were also enrolled for comparison. Blood samples were collected from patients and healthy donors into 5 mL vacutainer tube containing 3.5% buffered sodium citrate solution for measurement of total microparticles (TMPs), platelet microparticles (PMPs), endothelial microparticles (EMPs), CD62 activated platelets, and CD41 platelet marker.ResultsCOVID-19 malignant patients had significantly lower hemoglobin and platelets compared to COVID non-malignant ones, while they had significantly higher C-reactive protein, LDH, AST, Albunim, creatinine, and prognostic index (PI) compared to COVID-19 non-malignant patients. significant accumulations of TMPs, PMPs, EMPs, and activated platelets in COVID-19 affected patients compared to healthy controls. TMPs, and EMPs were significantly accumulated in COVID-19 malignant compared to COVID-19 non-malignant patients with no significant difference in PMPs between both.ConclusionCirculating MPs and activated platelets may be promising novel prognostic biomarkers capable of identifying potentially severe COVID-19 patients who require immediate care especially in cancer patients.

Pregnancy Alters Platelet Counts and Enhances Platelet Reactivity in Mice

Background: Pregnancy is a major risk factor for venous thromboembolism (VTE) and is the leading cause of maternal death during pregnancy and the immediate post-partum period. Although plasma levels of clotting factors and fibrinolysis inhibitors are known to increase during pregnancy peaking during the third trimester as parturition nears, little is known about whether platelets are affected in response to pregnancy. Platelets are derived from megakaryocytes and have a circulating half-life of 5-10 days. They are essential for blood coagulation, delivering procoagulant molecules directly to sites of vascular injury. Platelets contain a spliceosome that processes pre-mRNA, providing them with the necessary machinery to respond to external stimuli. Thus, platelets can be affected by diseases such as cancer, which can alter platelet mRNA profiles and function. The plasticity of platelets in these contexts suggests that they could also change in response to pregnancy as an adaptation to preventing blood loss during parturition. However, little is known about the transcriptional and/or functional changes that could occur in platelets during the course of pregnancy. Aims: We aimed to analyze mouse platelet function at early, middle, late gestation, and postpartum timepoints in order to identify pregnancy related functional changes. Methods: C57BL/6J female mice were mated with C57BL/6J male mice to generate pregnant mice. Pregnancy was confirmed by the presence of a vaginal plug, which was used to estimate embryonic day 0.5. Pregnant mice were randomly assigned to one of four groups based on gestational stages corresponding to human trimesters and postpartum. The stages are early (days 7-9), middle (days 11-14), late (days 16-20), and postpartum (days 22-24). The maximum volume of mouse whole blood was drawn into a 1:9 volume of buffered sodium citrate via cardiac puncture. A 20ul aliquot was used for complete blood counts using the Advia 2120 with settings optimized for C57BL/6 mouse blood. The remaining blood was reserved for platelet aggregation studies using the Roche Multiplate Aggregometer. ADP and type 1 collagen were used as the aggregating agents with the operator blinded to the gestational groups. Studies were initiated at the same time of day in order to control for the circadian rhythm and the gestational age. Results: The maximum blood volume we were able to draw from each mouse was significantly increased for all groups (early, middle and late gestational stages and postpartum) compared to the non-pregnant control group (p ≤ 0.03, n ≥ 9). Platelet counts (per microliter of blood) were statistically unchanged among the control and the three gestational groups. However, postpartum platelet counts were significantly increased relative to all groups (control, early, middle and late gestational stages) (p ≤ 0.02, n ≥ 9). There was a more vigorous platelet aggregation response in the late gestational group compared to the non-pregnant control and early gestational groups (areas under the curve with ADP (p ≤ 0.004, n ≥ 7) and collagen (p ≤ 0.028, n ≥ 7). Conclusions and future directions: Our results demonstrate significant changes in blood volume and a dramatic increase in postpartum platelet counts. Our results are consistent with those observed in pregnant humans, indicating that platelet counts have a similar response to pregnancy in both humans and mice. Our results demonstrating a late gestational increase in platelet reactivity suggest that pregnancy induced platelet alterations could play a role in the increased risk of VTE during pregnancy and the immediate post-partum period. We are currently performing additional analyses including RNA sequencing, to determine the mechanisms responsible for the phenotypic changes that we have observed here. The results of our studies could lead to enhanced treatment strategies for pregnancy associated VTE. Disclosures No relevant conflicts of interest to declare.

PRP Plasma Gel Protocolo de Obtenção para uso em procedimentos estéticos

O objetivo do presente artigo foi apresentar um protocolo de obtenção de derivados plaquetários que permite a sua utilização em procedimentos estéticos faciais. Métodos: O Plasma Rico em Plaquetas (PRP) foi obtido a partir da coleta de sangue em 6 tubos de 10 ml BD Vacutainer® de Citrato de Sódio Tamponado 0,109 mol/L que foram centrifugados a 150G por 15 minutos. A seguir foi coletada a porção amarela de cada tubo e o conteúdo obtido dividido em dois tubos (Tubo Vacuette®, 9 ml, Greiner Bio-One International, sem aditivos) que foram centrifugados novamente a 300 g por 5 minutos. Uma porção do plasma de 60% do volume do tubo foi aspirada em seringa de 3 ml e colocada em um aquecedor de seringas (APAG, Silfradent) a 75ºC por 15 minutos para obtenção do gel de plasma. A porção restante foi gentilmente agitada para ressuspender as plaquetas no plasma restante formando assim Plasma Rico em Plaquetas concentrado. O PRP Plasma Gel foi obtido mesclando o Gel de Plasma aquecido com o concentrado de PRP na proporção de 1:1, 2:1 e 3:1 para conseguir densidades diferentes de gel adequadas a indicação estética. Discussão: O PRP Plasma Gel produzido usando o protocolo 5G é uma alternativa prática ao uso de preenchedores comerciais em algumas áreas faciais. Os fatores de crescimento do concentrado plaquetário, seus fatores angiogênicos e a fibrina promovem o rejuvenescimento do tecido, reparando as fibras e células perdidas pelo envelhecimento. Conclusão: O uso do PRP Plasma Gel obtido pelo protocolo proposto é um procedimento simples, seguro e acessível a um maior número de pacientes promovendo uma melhora progressiva da pele.

DIPPING SOLUTIONS OF NISIN AND BUFFERED SODIUM CITRATE SUPPLEMENTED WITH SODIUM DIACETATE FAIL TO PREVENT GROWTH OF LISTERIA MONOCYTOGENES ON VACUUM PACKAGED BEEF FRANKFURTERS STORED AT 4 AND 10°C UNDER MODEL CONDITIONS

This study evaluated commercially available dip solutions of nisin alone, buffered sodium citrate combined with sodium diacetate (BSCSD), and combined solution of the three antimicrobials for potential to control growth of surface-inoculated Listeria monocytogenes during vacuum packaged refrigerated storage of model beef frankfurters. None of the treatments prevented growth of L. monocytogenes during Frankfurter storage. The combined treatments slowed growth of L. monocytogenes better than individual treatments. Failure to completely eliminate L. monocytogenes on frankfurters or inhibit outgrowth during storage might be attributed to high initial L. monocytogenes inoculum levels, insufficient quantities of antimicrobials to interact with all the target cells, low nisin activity at high pH, or presence of resistant subpopulations, such as nisin-resistant strains. The model conditions used in the experimental setup, such as elimination of natural microbiota from frankfurters and nutrient-rich diluent used for Listeria introduction on the surface, could also contributed to enhanced survival and growth of the pathogen.

Age-Associated Changes of Coagulation Test and Coagulation Factor Activities in Healthy Chinese Children

Background: The concept of developmental hemostasis has been universally accepted. Plasma concentrations of many coagulation factors in childhood are significantly different from adults for both mean values and ranges of normal. Thus, an understanding of developmental hemostasis and the development of appropriate age-dependent reference ranges are pivotal for prevention, diagnosis, and treatment of hemostatic problems during childhood. However, no data of developmental hemostasis are available in healthy Chinese children. Methods: Coagulation data from children aged 0-18 years old receiving minor elective surgery in Chengdu Women and Children's Central Hospital, from Sep. 2017 to Feb. 2019 was collected, and patient medical records were reviewed. Ethical approval was obtained from the parents of all children and the study was approved by the Ethics Review Committee at Chengdu Women and Children's Central Hospital. To qualify for the study, enrolled subjects had to meet the following criteria: (1) Children aged 0-18 years receiving minor elective surgery.(2) No history of bleeding problems.(3)No family history of bleeding nor thrombosis .(4) No history of heart, lung, liver or kidney diseases, normal physical examination. (5) Normal blood routine test. Normal liver function and kidney function, (6)No history of medication use for at least two weeks prior to specimen collection.(7) The operation gone successfully, and postoperative recovery was well. Blood samples (3 mL) were obtained by venipuncture into plastic tube containing 3.2% (109 mmol/L) buffered sodium citrate (1 part citrate:9 parts blood). The specimens were centrifuged immediately at 2500 g for 15 min at room temperature to prepare platelet-poor plasma (PPP) for the preoperative coagulation screening tests, consisting of activated partial thromboplastin time (APTT), prothrombin time (PT)), international normalized ratio (INR), thrombin time (TT) and fibrinogen. Activity of coagulation factors VIII, IX, XI, XII,Ⅱ,Ⅴ,Ⅶ,Ⅹ were determined using one-stage clotting methods with respective factor-deficient plasma. Results: A total of 82 samples were collected, while only 67 met the enrollment criteria. Specimens with inappropriate ratio of blood to anticoagulant, hemolysis or abnormal values of coagulation tests (APTT, PT, TT, fibrinogen) were excluded from analysis. The total of 67 children (40 Male 27 female), with a median age of 2.0 years (range: 1month -14 years) were divided into three groups according to the age: &lt;1y group 15 cases, 1-5y group 45 cases, and &gt;6y group 7cases. (1) The values of APTT, PT, TT, and fibrinogen were (36.1±5.2) seconds, (11.1±0.85) seconds, (20.3±1.6) seconds, (2.4±0.89) g/L respectively. No significant differences were found between groups. (2) In all children ,the activities of factor VIII、IX、XI、XII、vWF、II、V、VII、X were(123.6±48.3)%、(75.9±16.9)%、(95.9±24.3)%、(43.7±16.3)%、(111.3±50.4)%、(90.2±14.0)%、(104.7±21.1)%、(81.6±19.1)%、(93.0±21.8)% respectively. Factor VIII and vWF activities were significantly higher than other factors, while factor XII activities were significantly lower than others. (3)Mean values of FII:C, FVIII:C, FIX:C, FXI:C were significantly lower in children below &lt;1year old group than those in 1-5y group. No significant difference were seen in FVII:C, FV:C, FX:C, FXII:C, vWF:C among three groups. Conclusions: Coagulation test is just a simple and easy screening test. Coagulation factor activities changed dynamically with age during childhood, especially the FII:C, FVIII:C, FIX:C and FXI:C. Physiological reference ranges for coagulation factor activities in Chinese children of different ages should be established in order to evaluate the children with congenital or acquired bleeding diseases correctively. Table Disclosures No relevant conflicts of interest to declare.

A New Test for the Detection of Direct Oral Anticoagulants (Rivaroxaban and Apixaban) in the Emergency Room Setting.

First-in-human, 91-patient, single-center retrospective pilot study. Emergency room. Adult patients admitted into the emergency department, which received any clinician-ordered coagulation test requiring a 3.2% buffered sodium citrate blood collection tube. None. Plasma samples from patients admitted to the emergency department were screened for the use of FXa-Is, including apixaban and rivaroxaban, within the past 24 hours using our new i-II-X microfluidic test. i-II-X results were then compared with results from conventional coagulation tests, including prothrombin time (PT) and international normalized ratio (INR), which were ordered by treating clinicians, and an anti-Xa assay for rivaroxaban. The i-II-X test detected DOACs in samples collected from the emergency department with 95.20% sensitivity and 100.00% specificity. Unlike PT and INR, i-II-X reliably identified patients who had prolonged clotting times secondary to the presence of a FXa-I. The i-II-X test overcomes the limitations of currently available coagulation tests and could be a useful tool by which to routinely screen patients for DOACs in emergency and critical care settings. Our new diagnostic approach is particularly relevant in clinical situations where medical records may be unavailable, or where precautions need to be taken prior to invasive interventions, such as specific reversal agent administration.

Preanalytical Issues in Hemostasis and Thrombosis Testing.

Hemostasis testing is critical to many hemorrhagic and thrombotic disorders, wherein laboratory diagnostics can provide critical information for diagnosis, prognostication, and therapeutic monitoring. Due to this crucial role in modern medicine, hemostasis tests should be carried out at their highest degree of quality, thus encompassing standardization and monitoring of all phases of the testing process. It is now clearly established that the preanalytical phase is the most critical and vulnerable part of the total testing process, since up to 70% of diagnostic errors are due to highly manual activities encompassing patient preparation and collection of biological samples, as well as handling, transportation, preparation and storage of blood specimens. Due to the peculiar sample matrix required for hemostasis testing (i.e., plasma anticoagulated with buffered sodium citrate), additional critical issues may impair the reliability of these tests. Therefore, this article aims to provide an updated overview of the most important preanalytical variables that may ultimately impair the quality of hemostasis and thrombosis testing.

Abstracts of CSCC Conference 2016

Thus study was aimed to investigate whether the mixing technique could influence the results of routine and specialized clotting tests on post-thawed specimens.The sample population consisted of 13 healthy volunteers. Venous blood was collected by evacuated system into three 3.5 mL tubes containing 0.109 mmol/L buffered sodium citrate. The three blood tubes of each subject were pooled immediately after collection inside a Falcon 15 mL tube, then mixed by 6 gentle end-over-end inversions, and centrifuged at 1500g for 15 min. Plasma-pool of each subject was then divided in 4 identical aliquots. All aliquots were thawed after 2-day freezing − 70 °C. Immediately afterwards, the plasma of the four paired aliquots were treated using four different techniques: (a) reference procedure, entailing 6 gentle end-over-end inversions; (b) placing the sample on a blood tube rocker (i.e., rotor mixing) for 5 min to induce agitation and mixing; (c) use of a vortex mixer for 20 s to induce agitation and mixing; and (d) no mixing. The significance of differences against the reference technique for mixing thawed plasma specimens (i.e., 6 gentle end-over-end inversions) were assessed with paired Student's t-test. The statistical significance was set at p < 0.05.As compared to the reference 6-time gentle inversion technique, statistically significant differences were only observed for fibrinogen, and factor VIII in plasma mixed on tube rocker. Some trends were observed in the remaining other cases, but the bias did not achieve statistical significance. We hence suggest that each laboratory should standardize the procedures for mixing of thawed plasma according to a single technique.

Mixing of thawed coagulation samples prior to testing: Is any technique better than another?

ObjectiveThus study was aimed to investigate whether the mixing technique could influence the results of routine and specialized clotting tests on post-thawed specimens. MethodsThe sample population consisted of 13 healthy volunteers. Venous blood was collected by evacuated system into three 3.5mL tubes containing 0.109mmol/L buffered sodium citrate. The three blood tubes of each subject were pooled immediately after collection inside a Falcon 15mL tube, then mixed by 6 gentle end-over-end inversions, and centrifuged at 1500g for 15min. Plasma-pool of each subject was then divided in 4 identical aliquots. All aliquots were thawed after 2-day freezing −70°C. Immediately afterwards, the plasma of the four paired aliquots were treated using four different techniques: (a) reference procedure, entailing 6 gentle end-over-end inversions; (b) placing the sample on a blood tube rocker (i.e., rotor mixing) for 5min to induce agitation and mixing; (c) use of a vortex mixer for 20s to induce agitation and mixing; and (d) no mixing. The significance of differences against the reference technique for mixing thawed plasma specimens (i.e., 6 gentle end-over-end inversions) were assessed with paired Student's t-test. The statistical significance was set at p<0.05. Results and conclusionAs compared to the reference 6-time gentle inversion technique, statistically significant differences were only observed for fibrinogen, and factor VIII in plasma mixed on tube rocker. Some trends were observed in the remaining other cases, but the bias did not achieve statistical significance. We hence suggest that each laboratory should standardize the procedures for mixing of thawed plasma according to a single technique.

Effects of storage and thawing conditions on coagulation testing.

Current recommendations for coagulation testing storage and thawing are based on historical studies that were performed using unbuffered 3.8% sodium citrate. We sought to measure the effects of freezing and thawing conditions 3.2% buffered sodium citrate plasma samples that have been stored in vials with either snap or sealed screw tops, frozen in -70 °C freezer or dry ice and thawed either capped or uncapped. Shed blood samples were pooled and then aliquoted into four snap top and four screw tops vials. Half the vials were stored in a -70 °C freezer, and half on dry ice for at least 16 h. Afterwards, half the frozen samples were thawed in 37 °C waterbath capped, and other half were thawed capped. After thawing cycles, samples were tested for PT, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, factor assays, von Willebrand factor activity, plasminogen, antithrombin, protein C and lupus anticoagulant. Prothrombin time, APTT, factor X, and lupus anticoagulant testing were affected by all vials, freezing and thawing conditions, whereas fibrinogen, D-dimer, von Willebrand activity or protein C were not affected by any vial, freezing or storage condition. Storage vials, freezing and thawing condition affect coagulation testing, although these differences may not be clinically significant.

Systematic evaluation of evidence on veterinary viscoelastic testing Part 2: Sample acquisition and handling

To examine systematically the evidence on sample acquisition and handling for the thrombo elastography (TEG) and rotational thromboelastometry (ROTEM) viscoelastic point of care instruments and to identify knowledge gaps. Six questions were considered, addressing sampling site, collection system, anticoagulant, collection procedure, and sample storage. Standardized, systematic evaluation of the literature was performed. Relevant articles were categorized according to level of evidence (LOE). Consensus was developed regarding conclusions for application of concepts to clinical practice. Academic and referral veterinary medical centers. PubMed and CAB abstracts were searched. Eighteen papers were initially chosen; 5 of these papers applied to > 1 domain question. Three papers were used to address 2 questions each, and 2 papers were used to address 3 questions each. Most papers were judged LOE 3 (Good or Fair). Two of 5 papers were judged to be the same LOE each time they were used; 2 papers were judged to be LOE 3, Fair for 1 question and 3, Good for a second question; 1 paper used to address 3 questions was judged LOE 3, Good twice and 3, Fair once. Fourteen additional papers were evaluated post hoc during manuscript preparation. Jugular venipuncture is recommended, but samples from IV catheters can be used. Consistent technique is important for serial sampling, and standardized sampling protocols are recommended for individual centers performing TEG/ROTEM. There is insufficient evidence to recommend use of a specific blood collection system, although use of evacuated blood tubes and 21-Ga or larger needles is suggested. Use of 3.2% buffered sodium citrate in a strict 1:9 ratio of citrate to blood is suggested. Suggested tube draw order is discard/serum, followed by citrate, EDTA, and then heparin. Samples should be held at room temperature for 30 minutes prior to analysis.

The order of draw: myth or science?

The potential for cross-contamination of additives among evacuated blood tubes has led to the development of the order of draw. This practice, however, is mainly based on scarce, anecdotal, and mostly outdated literature data. Therefore, the goal of this investigation was to definitely establish whether or not the indication of a specific order of draw is still justified. The study population consisted of 57 outpatients referred to the outpatient oral anticoagulant (OA) clinic of the Academic Hospital of Verona and 58 healthy volunteers enrolled from the laboratory personnel. In OA outpatients, one serum tube was collected immediately after needle insertion, followed by a buffered sodium citrate tube and another serum tube. In the healthy volunteers, one serum tube was collected immediately after needle insertion, followed by a potassium-ethylenediaminetetraacetic acid (K2-EDTA) tube and another serum tube. After separation, the serum was tested for potassium, sodium, calcium, magnesium, and phosphorus in the first and second serum tubes. No significant difference could be observed between the first and the second serum tubes for any of the parameters. The bias calculated with Bland-Altman plots did not achieve statistical significance when the serum tube was collected after either a K2-EDTA or a sodium citrate tube. According to our data, revision of national and supranational recommendations on blood collection by venipuncture should consider that the order of draw exerts a negligible effect on sample quality, and this aspect should no longer be considered a quality criterion when evaluating the performance of phlebotomists.

Pre-analytical issues in the measurement of circulating microparticles: current recommendations and pending questions

Pre-analytical issues in the measurement of circulating microparticles: current recommendations and pending questions

Effects of vigorous mixing of blood vacuum tubes on laboratory test results

ObjectiveTo evaluate the effect of tubes mixing (gentle vs. vigorous) on diagnostic blood specimens collected in vacuum tube systems by venipuncture. Design and methodsBlood was collected for routine coagulation, immunochemistry and hematological testing from one hundred volunteers into six vacuum tubes: two 3.6mL vacuum tubes containing 0.4mL of buffered sodium citrate (9NC) 0.109mol/L: 3.2W/V%; two 3.5mL vacuum tubes with clot activator and gel separator; and two 3.0mL vacuum tubes containing 5.9mg K2EDTA (Terumo Europe, Belgium). Immediately after the venipuncture all vacuum tubes (each of one additive type) were processed through two different procedures: i) Standard: blood specimens in K2EDTA- or sodium citrate-vacuum tubes were gently inverted five times whereas the specimens in tubes with clot activator and gel separator were gently inverted ten times, as recommended by the manufacturer; ii) Vigorous mix: all blood specimens were shaken up vigorously during 3–5s independently of the additive type inside the tubes. The significance of the differences between samples was assessed by Student's t-test or Wilcoxon ranked-pairs test after checking for normality. The level of statistical significance was set at P<0.05. ResultsNo significant difference (P<0.05) was detected between the procedures for all tested parameters. Surprisingly only a visual alteration (presence of foam on the top) was shown by all the tubes mixed vigorously before centrifugation (Fig. 1 A, B and C). Moreover the serum tubes from vigorous mixing procedure shows a “blood ring” on the tube top after stopper removal (Fig. 1 D). ConclusionOur results drop out a paradigm suggesting that the incorrect primary blood tubes mixing promotes laboratory variability. We suggest that similar evaluation should be done using other brands of vacuum tubes by each laboratory manager.

EDTA-dependent pseudothrombocytopenia: further insights and recommendations for prevention of a clinically threatening artifact

Ethylenediaminetetra-acetic acid (EDTA) is widely used as anticoagulant in laboratory medicine. EDTA-dependent pseudothrombocytopenia is a rare phenomenon (i.e., around 0.1% in the general population), which is mostly due to the presence of EDTA-dependent antiplatelet antibodies that react optimally between 0°C and 4°C, recognize the cytoadhesive receptors gpIIb-IIIa, stimulate the expression of activation antigens, trigger activation of tyrosine kinase, platelet agglutination and clumping in vitro, which finally lead to a spuriously decreased platelet count. The reliable and timely identification of this artifact is essential, since there a high chance that it may be confused with other life-threatening platelet disorders, or otherwise lead to inappropriate clinical and therapeutic decision-making. Five basic criteria should be fulfilled to raise the clinical suspicion of EDTA-dependent pseudothrombocytopenia, i.e., (i) abnormal platelet count, typically <100×10(9)/L; (ii) occurrence of thrombocytopenia in EDTA-anticoagulated samples at room temperature, but to a much lesser extent in samples collected with other anticoagulants and/or kept warmed at ~37°C; (iii) time-dependent fall of platelet count in the EDTA specimen; (iv) evidence of platelet aggregates and clumps in EDTA-anticoagulated samples with either automated cell counting or microscopic analysis; (v) lack of signs or symptoms of platelet disorders. Several remedies have been proposed, such as warming the sample to 37°C or using additives or specific formulations of anticoagulants including buffered sodium citrate, heparin, ammonium oxalate, β-hydroxyethyltheophylline, sodium fluoride, CPT (trisodium citrate, pyridoxal 5'-phosphate and Tris), antiplatelet agents, potassium azide, amikacin, kanamycin or other aminoglycosides, and calcium replacement with the simultaneous addition of calcium chloride/heparin. According to available evidences, the most suitable and practical approach so far for most clinical laboratories seems, however, the recollection of blood samples using sodium citrate, CPT or calcium chloride/heparin as additives, maintaining the specimen at 37°C until the platelet count has been completed.

Comparison of plasma and peritoneal indices of fibrinolysis between foals and adult horses with and without colic

To identify hemostatic imbalances indicative of an increased risk of intra-abdominal adhesion formation in foals versus adult horses. Horses with colic undergoing exploratory laparotomy or abdominocentesis as part of a clinical examination (n = 16 foals ≤ 6 months of age and 19 adults ≥ 5 years of age) and horses without colic undergoing herniorrhaphy (15 foals) or euthanasia for noninflammatory and nongastrointestinal disease (10 foals and 20 adults). Paired abdominal fluid and blood samples were collected from each horse into buffered sodium citrate and centrifuged immediately after collection. Supernatants were stored at -80°C, then thawed for measurement of fibrinogen concentration, plasminogen activity, antiplasmin activity, and D-dimer concentration. Supernatant analyte concentrations or activities were compared within age group (foals with and without colic vs adults with and without colic) and within disease status (foals and adults without colic vs foals and adults with colic). All analyte concentrations or activities in abdominal fluid samples were significantly lower in the noncolic groups than in the colic groups, and none differed between foal and adult groups. Several plasma analyte values differed by disease status and age. The risk of intra-abdominal adhesion formation in the foals in this study did not appear to be attributable to differences in intra-abdominal hemostasis between adult horses and foals. Strategies for initial medical and surgical management of colic in adult horses may be applicable to foals with similar disorders.

P-selectin glycoprotein ligand-1 positive microparticles in allogeneic stem cell transplantation of hematologic malignancies

P-selectin and its receptor P-selectin glycoprotein ligand-1 (PSGL-1) mediate adhesion between leukocytes, tumor cells (including leukemias and lymphomas), and platelets, and play an important role in hematopoiesis, T cell activation, and cancer growth and metastasis. As microparticles (MPs) are released from activated or apoptotic cells, there should be significant numbers of circulating PSGL-1-bearing MPs in the blood of patients undergoing allogeneic stem cell transplantation (alloSCT). We enumerated PSGL-1-expressing MPs in plasma samples from 30 consecutive patients with hematologic disorders at different time points during the course of alloSCT by flow cytometry and analyzed their relation to cell counts, patient characteristics, and clinical outcome. Median follow-up time of surviving patients was 1,772 days (range 1272-1981 days). Nineteen patients (63.3%) died, 10 due to progression of disease (33.3%). The PSGL-1 MPs significantly declined during conditioning therapy but increased again after transfusion of donor cells and even more at the time of engraftment. Numbers >250/μL after graft transfusion were associated with a shorter time to engraftment for patients receiving fresh peripheral stem cell grafts (median, 15 vs. 21days; p = 0.049). Furthermore, low PSGL-1 MP values at those two time points were associated with a higher risk of progress/relapse in univariate analysis (p = 0.008-0.014; hazard ratio [HR] = 15.0-42.0) with cumulative incidences at 5 years of 81.8% versus 28.6% and 85.7% versus 20.0%, respectively. In conclusion, PSGL-1 microparticles show a characteristic course during alloSCT and their possible association with relapse/progress requires further evaluation of the PSGL-1/P-selectin interaction in leukemias and lymphomas.

Erythrocytapheresis in Sickle Cell Disease Lead to An Elective Reduction of Erythrocytes-Derived Blood Microparticles

AbstractAbstract 2657 Introduction:Erythocytapheresis is a highly effective procedure used in the management of both acute and chronic complications of sickle cell disease (SCD). Indeed, exchange transfusion is recommended as a part of the initial treatment of vaso-occlusive stroke in children. Red cells exchange can also be proposed in primary and secondary stroke prevention in order to maintain a low level of HbS. Microparticles (MPs) are small vesicles released from cells after activation or apoptosis. Microparticles number is increased in SCD both at the steady state and during crisis. The majority of these microparticles are originated from erythrocytes and platelets. In SCD, erythrocytes MPs are strongly correlated with hemolysis and activation of coagulation.In this study, we aimed to measure the level of total, platelets-derived and erythrocytes-derived MPs in patients with SCD undergoing erythrocytapheresis; we compared the level of MPs before and after red cells exchange in order to evaluate if the reduction of stroke observed in patients undergoing erythrocytapheresis could be due to a specific decrease in erythrocytes-derived MPs known to be implicated in coagulation activation in this disease. Materials and Methods:After informed consent, 24 samples were harvested on a tube containing buffered sodium citrate from 22 patients with SCD undergoing erythrocytapheresis. For each patient, a sample was collected before and just after the exchange. 20 samples from donor with neither anemia nor coagulation disorders were used as controls. Each sample was centrifugated at 1500g during 15 mn, the supernatant was collected and centrifugated 2mn at 13000g at 20°C and then frozen at -80°C until analysis. For MPs detection, 38mL of the thawed supernatant was labelled with 5mL of 10X Annexine V buffer, 1mL of Annexine V-FITC, 1 mL of anti-GPA-PE and 5 mL of anti-CD41-APC. 5mL of calibrated beads at known concentration were added to 345 mL of 1X Annexine buffer just before analysis on the MoFlow cytometer (Beckman Coulter). The Megamix beads system was used in order to delimitate the FSC/SSC gate of MPs. Results:Total number of plasma MPs (1755/mL vs 273), erythrocytes-derived MPs (174/mL vs 8) and platelets-derived MPs (1520/mL vs 236) were significantly increased in SCD patients in comparison with controls. For SCD patients undergoing erythrocytapheresis, the mean HbS level was respectively 52% and 24% before and after red cells exchange. No significant difference in the total hemoglobin concentration was noted. The mean number of MPs did not differ before and after the exchange (1755/mL versus 1940/mL respectively, student test p>0.05). The mean number of platelets-derived MPs remained stable before and after the exchange (1520/mL versus 1250mL, student test, p>0.05). In sharp contrast, we observed a significant clearance of the erythrocytes-derived MPs in the plasma of SCD patients after red cells exchange (mean number of GPA+-MPs: 174/mL before exchange versus 77mL after, student test, p<0.05). However, we could also observe a great variation of the number of erythrocytes and platelets-derived MPs between the different patients. Conclusion:The number of erythrocytes-derived MPs decreased specifically after red cells exchange, in contrast with the total and the platelets-derived MPs which remained stable; this selective decrease in the GPA+-MPs of SCD patients could be due to a reduction in the total number of red cells containing a high HbS content in the circulation, because of their replacement by normal erythrocytes that have a far lower capacity to produce MPs. It has been recently shown that in SCD, GPA+-MPs are specifically correlated with coagulation and fibrinolysis activation, underlying the fact that a direct link exists between hemolysis, erythrocytes- derived MPs liberation and procoagulant state. The specific clearance of the GPA+-MPs after red cells exchange could partially explain the positive effects of this procedure in primary and secondary prevention of stroke in SCD. However, that GPA+-MPs remained statistically increased after red cells exchange in comparison with controls; whether a low number of post-exchange MPs could be correlated with a better outcome is actually under investigation. Disclosures:No relevant conflicts of interest to declare.

Antimicrobial Effect of Buffered Sodium Citrate (BSC) on Foodborne Pathogens in Liquid Media and Ground Beef

The antimicrobial effects of a commercially available, buffered sodium citrate (BSC) were evaluated for the reduction of total aerobic bacteria count, Salmonella Typhimurium, Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus in a liquid medium and ground beef. BSC at 0, 1, 2 and 4.8% (wt/vol) or 0, 3, and 4.8% (wt/wt) was mixed into inoculated brain heart infusion (BHI) broth and ground beef (80% lean), respectively. BSC at concentrations of 1 and 2% did not inhibit growth of the pathogens tested in BHI broth. E. coli O157:H7 in BHI broth with 4.8% BSC was significantly reduced (p＜0.05) by 3～4 log CFU/mL compared with the control for up to 4 days. At 4.8%, BSC treatment of ground beef most significantly reduced (p＜0.05) total aerobic count and E. coli O157:H7 by 2.1 and 2.0 log CFU/g, respectively. This study indicates that the legally allowable level of 1.3% (wt/wt) BSC is not effective for reducing the pathogens tested in ground beef stored at 7℃.