Citrated Samples Research Articles

Rotational thrombolelastometry produces potentially clinical useful results within 10 min in bleeding Emergency Department patients

For the first time in the Emergency Department (ED), to assess the use of rotational thromboelastometry (ROTEM) in patients presenting with all-cause haemodynamic shock, specifically (a) to establish whether a 5- min (A5) or a 10-min result (A10) is accurate compared with a final maximum clot firmness (MCF) result; (b) to compare time to A10 and formal laboratory coagulation result; (c) to assess whether bleeding ED trauma, gastrointestinal and aortic aneurysm patients are coagulopathic according to ROTEM; and (d) to compare ROTEM results with formal laboratory coagulation parameters. Patients presenting to the ED in haemodynamic shock were recruited. A citrated coagulation sample was taken and once a ROTEM researcher arrived in the ED, was subjected to ROTEM analysis. Between 28 September 2010 and 31 August 2011, 40 patients were recruited (15 gastrointestinal bleeds, 20 major trauma cases and five ruptured abdominal aortic aneurysms). A10 and MCF correlated well (κ=0.98); A5 and MCF correlated less well (κ=0.91). The mean time to result (SD) was 57 (28) min for the formal laboratory coagulation result and 50 (45) min for the ROTEM A10 result (including delay to start of analysis). Seven patients were coagulopathic on ROTEM. Eighteen percent of bleeding ED patients are coagulopathic using ROTEM including 25% of trauma patients. A 10-min ROTEM clot firmness (A10) is an excellent surrogate for MCF and allows a result to be obtained earlier than formal laboratory results and potentially within 10 min of the patient arriving in the ED.

Coagulation Assessment in Children With Diabetic Ketoacidosis*

To assess potential hypercoagulability during diabetic ketoacidosis in children. A prospective, controlled pilot study. University-affiliated pediatric critical care unit and emergency department in a tertiary care children's hospital. Children (1-18 years) admitted with an episode of diabetic ketoacidosis and healthy children as controls. All patients with diabetic ketoacidosis managed according to a preestablished protocol. Thromboelastography was performed using citrated whole-blood samples drawn at the time of admission and upon biochemical and clinical resolution of diabetic ketoacidosis. Citrated whole-blood samples drawn from healthy nondiabetic children acted as control samples. Fifteen patients (11.7 ± 4.1 years) in the diabetic ketoacidosis group and 20 patients (8.9 ± 4.5 years; p = 0.06) in the control group completed the study. Values for standard thromboelastography parameters (R and K time, α angle, maximum amplitude, coagulation index, and Ly30) in the diabetic ketoacidosis group, both on admission and resolution, were within the control range; thromboelastography profiles of diabetic ketoacidosis patients on admission were not significantly different from profiles obtained upon diabetic ketoacidosis resolution. The mean α-angle was significantly higher in known diabetic patients compared with newly diagnosed diabetics on admission; however, it still remained within the control normal range. Thromboelastographic assay results do not reflect a hypercoagulable state in this group of children with diabetic ketoacidosis. Further investigation is required to examine the potential role of injured endothelium in the suspected hypercoagulability during diabetic ketoacidosis.

Basic Data for Reference Intervals in Koreans for Parameters Produced by Multiplate Platelet Function Analyzer

This article is available from http://www.labmedonline.org 2013, Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Background: The Multiplate analyzer (Dynabyte GmbH) has been recently introduced as a platelet function test for patients taking antiplatelet drugs. The study aimed at providing basic data for determining the reference interval of parameters produced by Multiplate in Koreans and to study the factors that influence those parameters. Methods: Blood was collected from 35 healthy volunteers (female 18, male 17) into tubes containing hirudin or 3.2% sodium citrate. Whole blood platelet aggregations triggered by adenosine-5’-diphosphate (ADP), ADP-high sensitive (ADP+PGE1 only in hirudin samples), arachidonic acid (AA), collagen or thrombin receptor activator peptide (TRAP) were investigated using Multiplate according to the manufacturer’s instructions. Data from healthy volunteers for the area under the curve (AUC) were determined from the central 95th percentile of the results. Results: The values of AUC in hirudin samples for all agonists were significantly higher than those in sodium citrate samples. The AUC values in hirudin (sodium citrate) samples were as follows: ADP 38-107 (18-119) U; ADP+PGE1 16-91 U; AA 64-156 (32-117) U; collagen 53-112 (26-108) U; and TRAP 81-163 (49-149) U. The parameters from Multiplate were significantly correlated with leukocyte counts, but not with hematocrit levels. Conclusions: Although our data were derived from only 35 subjects, the results are expected to be helpful in determining the reference interval at a single institute and may serve as basic data for future cumulative data of reference intervals from multiple institutes in Korea.

Effects of rest temperature, contact activation, and sample technique on canine thrombelastography

To determine the effects of rest temperature, contact activation (CA), and sample collection technique on thrombelastography (TEG) using canine whole blood. Prospective, experimental study. University-based research facility. Twelve healthy, adult, mixed-breed dogs. Blood was collected by jugular venipuncture. Tubes containing 3.2% sodium citrate, with and without 75 μg/mL corn trypsin inhibitor (CTI), were filled by vacuum. Samples rested for 30 minutes at 3 temperatures: 37°C, room temperature (RT, 20-22°C), or warmed to 37°C 5 minutes prior to analysis (prewarmed). Samples were analyzed at 37°C. CTI-treated samples were analyzed with and without 1:50,000 tissue factor (TF) as activator. Six dogs were also tested similarly using a needle/syringe collection technique. Prewarmed samples exhibited greater MA compared to RT (55.5 ± 7.2 mm vs. 53.5 ± 6.0, P< 0.05), while 37°C samples exhibited a steeper angle (56.7 ± 10.4°C vs. 52.4 ± 8.6°C) and greater MA (55.9 ± 7.5 mm vs. 53.5 ± 6.0 mm) than RT samples (both P< 0.05). CTI-treated samples were hypocoagulable (R time 45 min [7.5-56.8 min], angle 8.2°C [5.1-42.5°C], MA 29.2 ± 9.7 mm, P< 0.001), with TF activation returning all but the angle (42.5 ± 7.6°C) to values similar to citrated samples (angle = 56.7 ± 10.4°C, P = 0.017). Collection using a syringe/needle method revealed a shorter R time for prewarmed samples only (R time 4.7 ± 0.7 min, vs. 5.6 ± 0.8 min for vacuum-collected samples, P = 0.008). Even in the absence of exogenous activators, CA has an impact on canine TEG results. The effects of rest temperatures and sample collection technique on TEG appear to be minimal.

Temporal changes in ROTEM®-measured coagulability of citrated blood samples from coagulopathic trauma patients

BackgroundRotational thromboelastometry (ROTEM®) relies on citrated blood samples, which are regarded as biologically stable for up to 4h after venepuncture. However, this recommendation is based on data from normal volunteers. The aim of this study was to evaluate possible temporal changes in the coagulability of blood samples from coagulopathic trauma patients. Patients and methodsThis is a prospective series of 10 coagulopathic (maximum clot firmness, MCF<40mm) trauma patients. ROTEM® EXTEM (tissue factor activated) and FIBTEM (tissue factor activated, cytochalasin D inhibited) analyses were performed on samples obtained on admission, and after approximately 60min of storage in an incubator, at 37°C. ResultsThere were statistically significant differences between the median EXTEM MCF (22mm vs 54mm, p<0.001) and α angle (30.5 vs 59.5°, p=0.004) of the analyses performed immediately after sampling, and 51min (median) subsequently, but not coagulation time (CT, p=0.133), clot formation time (p=0.0625) or maximum lysis (ML, p=0.154). There were also no differences in median FIBTEM MCF (p=1.00) or CT (p=0.877) between the immediate and delayed analyses. ConclusionsRepeated ROTEM® EXTEM analysis of citrated samples from coagulopathic trauma patients shows a spontaneous improvement in coagulability with time. The absence of parallel changes on FIBTEM analysis suggests that this effect may be due to a change in platelet function.

In-vitro hypocoagulability on whole blood thromboelastometry associated with in-vivo expansion of red cell mass in an equine model

In several species, there is a strong correlation between indicators of red cell mass (RCM) and thromboelastometry results. The horse has a reliable, temporary, polycythemia in response to phenylephrine infusion. Our objective was to evaluate the effects of an in-vivo increase in circulating RCM on thromboelastometry results in an equine model of transient polycythemia. Six healthy research horses had whole blood thromboelastometry with contact activator and tissue factor initiation after recalcification of citrated samples. Additional samples were frozen for thrombin-antithrombin (TAT). Complete blood count biochemical analysis, fibrinogen, activated partial thromboplastin time (aPTT), and prothrombin time (PT) were performed. Additional samples were taken at 5 min and 2 h after phenylephrine infusion. Thromboelastometry was performed separately on four horses not receiving phenylephrine with the samples divided and spiked with phenylephrine ex vivo. Red cell count (P<0.001) and hematocrit (P<0.001) were significantly higher at 5 min after phenylephrine compared with baseline and 2 h. There was no change in platelet count, fibrinogen, PT, aPTT, or TAT at any time point. Both ex-tem and in-tem parameters were hypocoagulable at 5 min after phenylephrine compared to baseline and 2 h. There was no effect of phenylephrine in the ex-vivo spiking studies on any of the thromboelastometry parameters. Whole blood thromboelastometry results were hypocoagulable in this equine model of in-vivo transient polycythemia only during the polycythemic phase. All other coagulation parameters were unchanged. In the absence of other indicators of hypocoagulability, this may point to an artifact of thromboelastometry. Alternatively, the data may reflect true in-vivo hypocoagulability in patients with increased circulating RCM.

Do We Need Time Adjusted Mean Platelet Volume Measurements?

Mean platelet volume (MPV) is associated with various diseases. Several authors reported anticoagulant and time dependency. Therefore, standardized laboratory methods are essential. The aim of this study was to standardize the MPV measurement. Blood was collected in potassium-ethylenediaminetetra-acid (EDTA) and sodium-citrate tubes. First, MPV and platelet count were determined every half hour for 4 hours in 20 healthy volunteers. The same parameters were acquired from a second group of 100 healthy donors. We measured at the point of highest stability determined in the first step and aimed to determine a reference range. Citrate samples revealed significantly smaller MPV (7.0 fL ± 0.69 standard deviation [SD]) than EDTA (8.0 fL ± 0.8 SD). Platelets swell until 120 minutes in EDTA and until 60 minutes in citrate. Mean platelet count changed significantly in citrate. In the second group, no inverse correlation between MPV and platelet count was seen. A reference range was calculated (EDTA, 7.2-10.8 fL; citrate, 6.1-9.5 fL). Platelets stored in citrate are significantly smaller compared to those stored in EDTA. Timing is important when measuring platelet volume. Optimal measuring time should be 120 minutes after venipuncture. For this we depicted a reference range. Platelet count is most stable in EDTA. There was no inverse relation between MPV and platelet count.

ORIGINAL ARTICLE: Comparison of a point of care device against current laboratory methodology using citrated and EDTA samples for the determination of D‐dimers in the exclusion of proximal deep vein thrombosis

D-dimer estimation is a routine part of diagnostic algorithms for the exclusion of venous thromboembolism (VTE). We evaluated a point of care device, Biosite Triage (Inverness Medical UK, Cheshire, UK) for the estimation of D-dimers in both samples taken into citrate and EDTA against our routine laboratory D-dimer (Liatest D-dimer, Diagnostica Stago, Reading, UK) performed on the STA-R Evolution. With informed consent, 102 consecutive patients presenting with possible deep vein thrombosis (DVT) were enrolled and D-dimers along with Wells scores and compression ultrasonography (CUS) were recorded. Using the manufacturers' recommended cut offs of 500 microg/l fibrinogen equivalent units and 400 microg/l for the Stago and Triage, respectively, sensitivity, specificity, positive and negative predictive values were calculated. These were 1.00, 0.42, 0.17, and 1.00 for the Triage machine using citrate samples, 1.00, 0.32, 0.14, and 1.00 using EDTA samples and 1.00, 0.29, 0.16, and 1.00 for the Stago Liatest assay, respectively. Three patients had significantly higher results for the Stago Liatest D-dimer assay compared with the Biosite Triage device although ultrasound scans were negative. The Biosite Triage D-dimer assay performed on either citrate or EDTA samples is comparable with the Stago Liatest laboratory D-dimer assay when used in conjunction with clinical pretest probability scoring and CUS for the exclusion of DVT.

Differentiation of Enzymatic from Platelet Hypercoagulability Using the Novel Thrombelastography Parameter Delta (Δ)

Thrombelastography (TEG) allows for rapid global assessment of coagulation function. Our previous work demonstrated that a hypercoagulable state identified by TEG's G value was associated with thromboembolic events in a cohort of critically ill surgical patients despite routine chemoprophylaxis. We hypothesized that a hypercoagulable state could be differentiated into enzymatic or platelet etiology through the use of thrombus velocity curves; specifically the time to maximum rate of thrombus generation (TMRTG) and the novel TEG parameter, delta. (Delta) We retrospectively studied 10 critically ill surgical patients receiving thromboprophylaxis for at least 72 h by TEG, using kaolin activated citrated samples. Thrombus velocity curves were plotted for each patient, and delta was calculated as the difference between the TEG parameters R and SP, corresponding to the time to maximum rate of thrombus generation (TMRTG), which reflects the enzymatic contribution to clot formation. The TEG parameter G, (G = 5000 x A/100-A) also was determined for each patient. As G is derived from amplitude (A), it reflects overall net clot strength. A hypercoagulable state was defined as delta < 0.6 min and/or G > 11 dynes/cm(2). A hypercoagulable state was identified via delta in 6 patients (60%); all of whom remained hypercoagulable following heparinase addition, suggesting chemoprophylaxis was ineffective. Of six patients with a hypercoagulable G value, 50% had a normal delta suggesting the presence of platelet hypercoagulability. Delta closely correlated with TMRTG (r = 0.94). However, the varying contribution of platelets to hypercoagulability, was shown by a nonlinear, weak correlation of delta and TMRTG with G (r = 0.11 and r = 0.14, respectively). Delta reflects changes in thrombin generation as measured by TMRTG, allowing for differentiation of enzymatic from platelet hypercoagulability. Future studies will be required to validate these findings.

Can Thromboelastography performed on kaolin‐activated citrated samples from critically ill patients provide stable and consistent parameters?

Thromboelastography (TEG) is a potentially useful tool but analysis within 4-6 min of collection imposes limitations on its use and access. The use of citrate blood tubes potentially increases the time frame for processing specimens. There is, however, limited research on the stability of citrate specimens, timing of processing and the accuracy of TEG results. The purpose of this study was to examine the effects of early and delayed processing on TEG parameters using kaolin-activated citrated blood samples in the intensive care population. TEG analysis was performed on 61 patients. Blood was collected into two 3.2% sodium citrate (0.105 m) tubes. Kaolin-activated samples were analysed at 15, 30 and 120 min postcollection. TEG parameters analysed included reaction time (R), clot formation time (K), alpha angle (alpha), maximum amplitude, LY30, the coagulation index, time to maximum rate of thrombus generation, maximum rate of thrombus generation and total thrombus generation. Sixty-one critically ill patients were included. The results of the anova showed that time from collection was significantly associated with the TEG((R)) results (P < 0.05). On comparison of individual outcome variables, this difference in most cases was due to changes over time from 30 to 120 min. Furthermore, progressive changes in TEG parameters such as decreasing R were suggestive of a trend toward hypercoagulability of the specimens. Processing of kaolin-activated citrate TEG specimens can begin as early as 15 min postvenipuncture. However, delaying processing by more than 30 min leads to a significant change in results.

Adaptive force sonorheometry for assessment of whole blood coagulation

Viscoelastic diagnostics that monitor the hemostatic function of whole blood (WB), such as thromboelastography, have been developed with demonstrated clinical utility. By measuring the cumulative effects of all components of hemostasis, viscoelastic diagnostics have circumvented many of the challenges associated with more common tests of blood coagulation. We describe a new technology, called sonorheometry, that adaptively applies acoustic radiation force to assess coagulation function in WB. The repeatability (precision) of coagulation parameters was assessed using citrated WB samples. A reference range of coagulation parameters, along with corresponding measurements from prothrombin time (PT) and partial thromboplastin time (PTT), were obtained from WB samples of 20 healthy volunteers. In another study, sonorheometry monitored anticoagulation with heparin (0-5 IU/ml) and reversal from varied dosages of protamine (0-10 IU/ml) in heparinized WB (2 IU/ml). Sonorheometry exhibited low CVs for parameters: clot initiation time (TC1), <7%; clot stabilization time (TC2), <6.5%; and clotting angle (theta), <3.5%. Good correlation was observed between clotting times, TC1 and TC2, and PTT (r=0.65 and 0.74 respectively; n=18). Linearity to heparin dosage was observed with average linearity r>0.98 for all coagulation parameters. We observed maximum reversal of heparin anticoagulation at protamine to heparin ratios of 1.4:1 from TC1 (P=0.6) and 1.2:1 from theta (P=0.55). Sonorheometry is a non-contact method for precise assessment of WB coagulation.

A Comparison Between the StaRRsed Auto-Compact Erythrocyte Sedimentation Rate Instrument and the Westergren Method

BackgroundThe Westergren method is the golden standard for measuring erythrocyte sedimentation rate (ESR). All ESR methods should agree with the standardized method of the International Council for Standardization in Hematology (ICSH). Citrate samples are commonly used for ESR. This extra sample adds costs and can be inconvenient for the patient. Therefore, some new automated ESR analyzers use EDTA samples, which are available for other hematology measurements.MethodsWe compared ESR measurements with StaRRsed Auto-Compact instrument to the ICSH standardized Westergren method in 200 patient samples.ResultsThe correlation between methods was fairly good (R2 = 0.72, y = 1.066x 0.24). However, with ESR results over 11 mm/h there were 55 subjects with a difference of over 30% between methods.ConclusionsThis may have led to different treatment suggestions in 25 cases according to age- and gender-dependent normal values. The difference may be caused by two different anticoagulants used, different measuring times and the correlation equation used. The StaRRsed ESR method should be in better agreement with the Westergren method, which is the golden standard. ESR results have notable impact on patient diagnosis and follow-up.KeywordsESR; Erythrocyte sedimentation rate; StaRRsed; Westergren method

Preliminary Performances of A NEW Generation Assay : VIDAS D-Dimer Exclusion II.

Abstract 4203The diagnostic value of D-dimer (DD) in the exclusion of deep venous thrombosis (DVT) and pulmonary embolism (PE) is well established. With more than 8,000 patients included in prospective management studies, VIDAS D-Dimer assays have been the most extensively assay studied. The aim of this study is to evaluate the analytical performances of a new generation of D-Dimer assay on the VIDAS instrument: VIDAS D-Dimer Exclusion II* (bioMérieux, Marcy l'Etoile, France). VIDAS D-Dimer Exclusion II (DEX2) assay principle combines a two-step enzyme immunoassay sandwich method with a final fluorescent detection. Total duration of the test is 20 minutes. Calibration is performed once every 28 days with one standard. Calibrator and controls (n=2) are reconstituted with distilled water. VIDAS D-Dimer Exclusion II has been calibrated against a panel of human plasma samples, the concentrations of which have been determined using the VIDAS D-Dimer Exclusion kit (DD2).Studies were conducted to verify analytical performances of the new assay. Precision studies were performed according to CLSI document EP5-A2 using five plasma samples covering the range of the assay. The total %CV observed was less than 7%, including for those values around the areas of clinical decision limits. Limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) were evaluated according to CLSI document EP17-A and they were found at 7.41, 16.47 and 29 ng/mL (FEU) respectively. Linearity from a range of 45 to 10,000 ng/mL (FEU) has been determined on plasmas according to the recommendations of CLSI document EP6-A. Deviation from linearity was found to be less than 11%. Studies were conducted to verify the performance of the assay on potentially interfering substances such as bilirubin, triglycerides, hemoglobin, human albumin, and 47 commonly used drugs and anticoagulants. Less than 10% or 12% interference was observed at 500 and 7500 ng/mL (FEU) or 250 ng/mL (FEU) levels respectively, for each interfering substance. The assay Reference Interval was determined by testing 215 citrated plasma samples obtained from healthy volunteers; 90% of those values were found to be below the clinical cut off of 500 ng/mL (FEU). A method comparison study was performed on 584 citrated samples according to the recommendations of CLSI document EP9-A2. Regression line between VIDAS D-Dimer Exclusion II (DEX2) and VIDAS D-Dimer Exclusion (DD2) was: DEX2 = 1.04 x DD2 – 20,74. Observed bias was –4.53%, -0.38% and 3.07% at 250, 500 and 3000ng/mL (FEU) respectively.In conclusion, VIDAS D-Dimer Exclusion II (DEX2) assay demonstrated good performance characteristics, particularly around the clinical cut off, as well as an excellent overall agreement with the extensively studied and published prior generation assay, VIDAS D-Dimer Exclusion (DD2). With the ability to provide results in 20 minutes and with optimized total precision, VIDAS D-Dimer Exclusion II assay (DEX2) will play a key role in the laboratory to provide accurate, rapid and reliable answers to clinicians.* Product not cleared for use in the USA Disclosures:No relevant conflicts of interest to declare.

Thromboelastographic Methods to Detect Hypercoagulability.

Abstract 4006 Poster Board III-942 Introduction Venous thromboembolic events (VTE) cause significant morbidity and mortality. Based on various studies, the incidence of VTE is greater than 275,000 new cases per year in the United States (Heit JA, J Thromb Haemost. 2005. 3:1611-17). A laboratory assay that predicts the development of VTEs in high-risk populations could have significant impact by potentially leading to prophylactic therapy and minimizing sequelae from such events. The thromboelastograph (TEG) is a point of care global hemostatic assay that utilizes whole blood to provide a continuous graphic record of the physical dynamics of a clot during fibrin formation and subsequent lysis. In general, the TEG has been used most extensively to predict bleeding in the settings of liver transplantation and cardiac bypass surgery. When employing the manufacturer's standard methods, the TEG is suited to demonstrate reduced clotting capability (“hypocoagulability”). Previous studies using the thromboelastograph to detect hypercoagulability have yielded inconsistent results as the methods and parameters studied have varied. We have developed a novel method whereby the baseline, i.e., normal, TEG curve and parameters, are “hypocoagulable,” i.e., similar to that of a hemophilia patient, making hypercoagulability easier to detect. Methods After assessing a variety of pre-analytical conditions including withholding activation with either kaolin, celite, or tissue factor, re-calcifying citrated samples with varying amounts of calcium chloride, and addition of varying amounts of corn trypsin inhibitor (CTI, an intrinsic pathway inhibitor), the following method performed best at achieving the desired hypocoagulable TEG curve and parameters. Whole blood was collected from healthy adult volunteers into 3.2% sodium citrate tubes that were pre-loaded with 300 micrograms of CTI. The blood was then placed into the TEG and re-calcified with calcium chloride (no activators were used), and the TEG was run until full clot formation. In order to demonstrate that this novel method is sensitive to increased thrombin generation (a surrogate marker for hypercoagulability), semi-logarithmic concentrations of recombinant human thrombin were added ex vivo to blood from healthy adult volunteers and the assays were again run until full clot formation. Data were collected and descriptive statistics calculated. Results With the methods described above we were able to consistently create TEG curves that were hypocoagulable as compared to standard methods (see table below). The TEG curves of our novel method showed prolonged clot initiation (increased r time), slower clot propagation (increased k time and decreased angle), and fairly similar maximum clot strength (MA). The addition of increasing concentrations of thrombin showed increasing hypercoagulability as measured by TEG parameters (decreasing r time, decreasing k time, increasing angle and MA) and curves compared to baseline. Conclusions The results indicate that with manipulation of the pre-analytic conditions, normal individuals can have a profoundly “hypocoagulable” TEG curve and parameters making the assay sensitive to detecting hypercoagulability. In addition, the results demonstrate the sensitivity of this assay to increasing concentrations of thrombin, a surrogate marker of hypercoagulability. Future studies will apply these methods to populations at high risk for VTE and will aim to predict VTE in those patients. Disclosures: No relevant conflicts of interest to declare.

Effect of anticoagulants on the plasma hyaluronidase activities

Mammalian hyaluronidases (HAases) are an endo-beta-N-acetyl-hexosaminidases that degrade hyaluronan (HA) and have been implicated in diverse pathophysiological functions. Several pathological conditions, such as diabetes, monoclonal gammapathy, and bladder and prostate tumors, report the distorted plasma HAase activity. However, the plasma HAase (hHyal-1) activity has been presumed to change with the circulating HA level and serves as an early marker for several diseases. It has been generally practised to use the anticoagulants such as tri-sodium citrate/di-sodium EDTA/heparin for the preparation of plasma for both biochemical and clinical analyses. In the present investigation, the effect of anticoagulants on plasma HAaseactivity was evaluated and compared with the serum HAase activity that is devoid of anticoagulants as no study provides information in this regard. The results suggested that the plasma HAase activity in the presence of the recommended concentration of EDTA was highly comparable/similar to that of the serum HAase activity. In contrast, citrated or heparinized plasma recorded a significantly reduced level of activity than that of the serum HAase activity. In conclusion, our results suggested that the EDTA-treated plasma samples are a better choice compared with heparin and citrated samples to assess the HAase activity.

Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean platelet volume

Platelet counts on whole blood samples collected into tripotassium salt of EDTA, trisodium citrate (Na3 citr), citrate phosphate dextrose adenine formula 1 (CPDA-1) and acid citrate dextrose formula A (ACD-A), all showed a statistically significant drop (P less than 0.01) after 1 h standing at room temperature (RT) as compared with the immediate (within 30 min) counts. After 1 h the enumeration became stable in the EDTA samples but the drop continued up to 4-6 h in those samples taken into citrate. The decreases in citrate were significant (18-30%, P less than 0.001). The addition of EDTA (1.5 mg/ml) to the citrated samples after the sixth hour count created a significant rise (6-22%, P less than 0.01) in the counts between the sixth and the seventh hour. Our observations show that platelet counts in citrated blood samples are lower than those in EDTA and highlight the necessity to present citrated samples mixed wtih dried EDTA when characterization or quality control of blood and blood components is required. Analysis of the mean platelet volume (MPV) showed significantly lower values (6-13%, P less than 0.05) in the citrated samples as compared to the same samples in EDTA, and a significant increase (4-6%, P less than 0.01) on the addition of EDTA to the citrated samples after the sixth hour analysis.

Technical report: Analysis of citrated blood with thromboelastography: comparison with fresh blood samples

Thromboelastography (TEG) evaluates the visco-elastic properties of whole blood to assess clot formation and hemostasis. When blood cannot be analyzed immediately, it is stored in citrated tubes to be analyzed after recalcification. In this study, we evaluated the results of TEG analysis performed on citrated blood and compared these results to values obtained from activated (kaolin and tissue factor) and non activated, fresh blood samples, obtained at various time intervals (one, two, and three hours). Four blood samples were collected from each of ten healthy volunteers. The following TEG analyses were performed on each sample: reaction time (r), k time (k), alpha angle (alpha), and maximum amplitude (MA). Studies were done using fresh, non citrated blood, obtained within five minutes of collection, and using citrated blood, one, two, and three hours after collection. Samples were analyzed, with and without activation, using kaolin and tissue factor. Tissue factor activated and non activated, citrated samples had shorter r and k times (P=0.03, P=0.008, P<0.0001, and P<0.0001, respectively) and higher alpha angle and MA values (P<0.0001, P<0.0001, P=0.79, and P=0.03, respectively) compared to fresh, non citrated samples. These findings were consistent with a hypercoagulable state. Conversely, citrated samples, activated with kaolin, yielded results similar to those obtained from fresh, non citrated samples. The TEG measurements were similar among citrated samples stored from one to three hours. Our results demonstrate that TEG measures, performed on citrated blood samples, yield results that are consistent with a hyperocoagulable state. Using kaolin to activate citrated samples, on the other hand, yields results similar to those obtained from non citrated, fresh blood samples.

High intensity ultrasound activation of coagulation factors

Although it has been demonstrated that High Intensity Focused Ultrasound (HIFU) can induce vascular cauterization, acoustic hemostasis technology has not been successful in making the transition from proof-of-concept to clinical settings. One reason for this lack of acceptance is the limited understanding of the fundamental mechanisms involved in ultrasound-vessel and ultrasound-blood interactions. The aim of this research was to investigate the hematological and biochemical mechanisms which are influenced by HIFU induced coagulation. HIFU was used to induce coagulation in an in vitro hematological flow system and in animal models. The flow circuit and in vivo arteries were instrumented with flow probes and thermocouples in the treatment region. Physiological parameters were recorded throughout the in vivo experiments. Blood samples were drawn for analysis prior to, during, and immediately following each HIFU treatment. Clotting time, complete blood count, and biochemical analysis were performed immediately and citrated samples were immediately centrifuged and frozen for future analysis. Results indicate that HIFU can change local and systemic levels of thombin/anti-thrombin complex (TAT) and tissue plasminogen activator (tPA), as well as induce changes in activated clotting time (ACT). These results indicate that HIFU can induce coagulation via the coagulation cascades (TAT) and that normal hematological response to thrombus formation is unaffected.

ADAMTS 13 in non‐thrombotic thrombocytopaenic purpura conditions

ADAMTS 13 in non‐thrombotic thrombocytopaenic purpura conditions

Comparison of ethylenediaminetetraacetic acid and sodium citrate as anticoagulants in collection of samples for cytomegalovirus pp65 antigen detection in renal transplant recipients with suspected cytomegalovirus disease

Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients. Antigenemia and polymerase chain reaction (PCR) assay are used for diagnosis of CMV disease. A number of anticoagulants are used for the collection of blood samples for antigenemia assay. Thus, ethylenediaminetetraacetic acid (EDTA) and sodium citrate are evaluated for the collection of blood samples and their effects on antigenemia and PCR. Twenty renal transplant recipients with clinically suspected CMV disease and 10 healthy individuals were included in the study. Peripheral blood mononuclear cells (PBMCs) extracted from blood samples were subjected for antigenemia and PCR assay. In 15 out of 20 patients, the number of peripheral blood mononuclear cells obtained were higher in EDTA anticoagulated samples than in sodium citrate. CMV pp65 antigenemia was detected in 10 EDTA and 9 sodium citrate samples, respectively. Number of antigen positive cells in EDTA samples were significantly higher than that of sodium citrate (P<0.05). None of the anticoagulants had adverse effect on the detection of CMV DNA. Thus, EDTA was found to be a better anticoagulant for separation of PBMCs and thus, for CMV pp65 antigenemia assay than sodium citrate.