Blood Heparin Research Articles

Inactivation of heparin by cationically modified chitosan.

This study was performed to evaluate the ability of N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), the cationically modified chitosan, to form biologically inactive complexes with unfractionated heparin and thereby blocking its anticoagulant activity. Experiments were carried out in rats in vivo and in vitro using the activated partial thromboplastin time (APTT) and prothrombin time (PT) tests for evaluation of heparin anticoagulant activity. For the first time we have found that HTCC effectively neutralizes anticoagulant action of heparin in rat blood in vitro as well as in rats in vivo. The effect of HTCC on suppression of heparin activity is dose-dependent and its efficacy can be comparable to that of protamine-the only agent used in clinic for heparin neutralization. HTCC administered i.v. alone had no direct effect on any of the coagulation tests used. The potential adverse effects of HTCC were further explored using rat experimental model of acute toxicity. When administered i.p. at high doses (250 and 500 mg/kg body weight), HTCC induced some significant dose-dependent structural abnormalities in the liver. However, when HTCC was administered at low doses, comparable to those used for neutralization of anticoagulant effect of heparin, no histopathological abnormalities in liver were observed.

Most Blood Biomarkers Related to Vitamin Status, One-Carbon Metabolism, and the Kynurenine Pathway Show Adequate Preanalytical Stability and Within-Person Reproducibility to Allow Assessment of Exposure or Nutritional Status in Healthy Women and Cardiovascular Patients1–3

Knowledge of stability during sample transportation and changes in biomarker concentrations within person over time are paramount for proper design and interpretation of epidemiologic studies based on a single measurement of biomarker status. Therefore, we investigated stability and intraindividual vs. interindividual variation in blood concentrations of biomarkers related to vitamin status, one-carbon metabolism, and the kynurenine pathway. Whole blood (EDTA and heparin, n = 12) was stored with an icepack for 24 or 48 h, and plasma concentrations of 38 biomarkers were determined. Stability was calculated as change per hour, intraclass correlation coefficient (ICC), and simple Spearman correlation. Within-person reproducibility of biomarkers was expressed as ICC in samples collected 1–2 y apart from 40 postmenopausal women and in samples collected up to 3 y apart from 551 patients with stable angina pectoris. Biomarker stability was similar in EDTA and heparin blood. Most biomarkers were essentially stable, except for choline and total homocysteine (tHcy), which increased markedly. Within-person reproducibility in postmenopausal women was excellent (ICC > 0.75) for cotinine, all-trans retinol, cobalamin, riboflavin, α-tocopherol, Gly, pyridoxal, methylmalonic acid, creatinine, pyridoxal 5′-phosphate, and Ser; was good to fair (ICC of 0.74–0.40) for pyridoxic acid, kynurenine, tHcy, cholecalciferol, flavin mononucleotide, kynurenic acid, xanthurenic acid, 3-hydroxykynurenine, sarcosine, anthranilic acid, cystathionine, homoarginine, 3-hydroxyanthranilic acid, betaine, Arg, folate, total cysteine, dimethylglycine, asymmetric dimethylarginine, neopterin, symmetric dimethylarginine, and Trp; and poor (ICC of 0.39–0.15) for methionine sulfoxide, Met, choline, and trimethyllysine. Similar reproducibilities were observed in patients with coronary heart disease. Thus, most biomarkers investigated were essentially stable in cooled whole blood for up to 48 h and had a sufficient within-person reproducibility to allow one-exposure assessment of biomarker status in epidemiologic studies. The Western Norway B Vitamin Intervention Trial was registered at clinicaltrials.gov> as NTC00354081.

Reappearance of Circulating Heparin in Whole Blood Heparin Concentration-Based Management Does Not Correlate With Postoperative Bleeding After Cardiac Surgery

The Hepcon Heparin Management System (HMS) facilitates administration of higher heparin and lower protamine doses, which may affect bleeding potential due to heparin rebound. The present study evaluated heparin rebound in patients for whom the Hepcon HMS was used to determine whether point-of-care tests detect residual heparin and residual heparin is associated with postoperative blood loss. Prospective study. Tertiary care center affiliated with a university hospital. Adults undergoing elective cardiac surgery requiring cardiopulmonary bypass. In blood samples obtained at baseline, at 2 minutes, and at 1, 2, 4, 6, and 24 hours after heparin neutralization, heparin concentrations were measured using an automated chromogenic assay. Activated coagulation time (ACT), activated partial thromboplastin time (APTT), and thromboelastometry 2 hours after heparin neutralization also were examined in the last 22 study patients enrolled. All 31 patients had measurable heparin levels 2 hours after protamine administration; 22 patients exhibited a primary failure to reverse heparin after protamine administration, and 9 patients had measureable heparin levels 2 hours after complete heparin reversal (ie, heparin rebound). The thromboelastometric variable, INTEM-CT:HEPTEM-CT ratio, correlated with heparin concentration (r=0.72), but ACT (r=-0.12), APTT (r=0.36), and whole blood heparin concentration, determined using the Hepcon HMS, did not. Peak heparin concentration (0.18±0.07 U/mL) at 4 hours was not correlated with mediastinal blood loss. Circulating heparin detected by the chromogenic assay was too low to be clinically significant based on postoperative bleeding, although all 31 patients had residual heparin or heparin rebound at 2 hours after protamine administration with use of the Hepcon HMS.

Thin Layer Coulometry Based on Ion-Exchanger Membranes for Heparin Detection in Undiluted Human Blood

We explore here for the first time a potentially calibration-free methodology for the detection of protamine (and, by titration, heparin) in undiluted human blood in the therapeutic concentration range from 20 to 120 mg L(-1). The use of a thin layer sample (5.8 μL) confined between a tubular protamine selective membrane (inner diameter, 600 μm) and a Ag/AgCl wire (diameter 400 μm) achieves an exhaustive depletion from the sample. Coulometry detection was chosen for the interrogation of the thin layer, employing a double pulse technique with 120 s for each pulse. Protamine calibration curves were recorded at physiological concentrations and in undiluted human blood. A linear relationship was obtained in both cases, but a diminished sensitivity was observed in contact with blood, which is explained with a partial passivation of the inner Ag/AgCl element. Heparin-protamine titrations were performed in undiluted human blood samples, mimicking the final application with patients undergoing critical care. The observed values correlate satisfactorily with those of an alternative technique, so-called flash-chronopotentiometry on planar membranes.

Thrombin based gelatin matrix and fibrin sealant mediated clot formation in the presence of clopidogrel

BackgroundPlatelet inhibitors are commonly used to reduce the risk of atherothrombotic events. The aim of this study was to determine the impact of platelet inhibitors, specifically clopidogrel and aspirin, on clot kinetics, strength, and/or structure during the use of thrombin based gelatin matrices and fibrin sealants.MethodsBlood was collected and heparinized from donors on clopidogrel (and aspirin) and age matched control donors. Blood component analysis, whole blood platelet aggregometry, and activated clotting time (ACT) were used to monitor compliance to therapy and identify any differences between donor groups. Clot kinetics and strength were analyzed using thrombelastography (TEG). Field Emission Scanning Electron Microscopy (FESEM) was used to analyze clot structure.ResultsBlood component profiles were similar for both donor groups. Aggregometry indicated that aggregation response to adenosine diphosphate (ADP) for clopidogrel donors was 12% of that for the controls (p = 0.0021), an expected result of clopidogrel induced platelet inhibition. However, blood from both donor groups had an elevated thrombin induced aggregation response. Heparinization of donor blood resulted in similarly elevated ACTs for both donor groups. TEG results indicated similar clot kinetics and strength between clopidogrel and control donor groups for blood alone and when clotting was induced using thrombin based gelatin matrices and fibrin sealants. FESEM images supported TEG findings in that similar morphologies were observed in ex vivo formed clots from both donor groups when thrombin based gelatin matrices and fibrin sealants were used.ConclusionThese results suggest that platelet inhibitors do not negatively impact clot kinetics, strength, and structure when clotting is initiated with thrombin based gelatin matrices and fibrin sealants.

Genetic analysis in a true hermaphrodite: A case report

Background: True hermaphrodite is one of the rarest variety of disorders of sexual differentiation (DSD) and represents only 5% cases of all. True hermaphroditism is characterized by the development of ovarian and testicular tissue in the same individual. Müllerian and Wolffian structures are usually present, and external genitalia are often ambiguous. The most frequent karyotype is 46, XX or various forms of mosaicism. We report a 8yrs old child of a serving soldier reared as male. He underwent orchidectomy for right undescended testis at 18 months of age. The biopsy of the testis showed presence of granulosa cells. The external genitalia were clearly identified as male penis with hypospadius. The patient reported to us for cyto-molecular analysis. Methods: 8ml of venous blood was collected, 5ml in heparin and 3ml in EDTA. The blood in heparin was processed for lymphocyte culture for 72h followed by Trypsin Giemsa banding of cells arrested in metaphase using standard protocol. Cytogenetic analysis of peripheral blood revealed mosaic karyotype with 90% 46, XX and 10% 47, XXY cell line. Genomic DNA was isolated from blood sample in EDTA and then subjected to PCR amplification of SRY gene which showed desired band at 500 bp (SRY gene). Conclusion: An early gender assessment of true hermaphrodites is of utmost importance for proper psychosocial development of child. Though phallic size is the single most important criteria for gender assessment, a multidisciplinary team of specialists in consultation with parents must assign sex of the child on basis of genitalia, gonads and genetic factors.

Clot-On-A-Chip: A Microfluidic Device To Study Platelet Aggregation and Contractility Under Shear

IntroductionIn primary hemostasis, platelets adhere, activate, and aggregate at the wall of an injured vessel to form a hemostatic plug for the cessation of bleeding. After activation, platelets generate myosin-driven contractile forces to compact the size of the plug in order to reduce the space between platelets and prevent their disaggregation. Hemodynamic shear can be a major effector of platelet function in hemostasis, but its effect on the ability of platelets to produce contractile forces is an open question. Studying the dynamics of platelet aggregation and platelet force generation under hemodynamic shear can provide important insights into hemostasis and thrombosis. MethodWe have developed a microfluidic device that uses microscale blocks to induce platelet aggregation and microscale posts to measure platelet forces in a hemostatic plug. Whole human blood in heparin or citrate is pumped through a microfabricated chip containing microchannels with arrays of blocks and posts arranged along the bottom of a microchannel (Fig. 1). The surface of the blocks and posts are pre-coated with von Willebrand factor and type I collagen to allow for platelet adhesion. As blood is passes over a block, its rectangular shape induces a high shear rate that causes platelets to aggregate on its surface. A flexible micropost is situated behind each block. As platelets aggregate between the block and post, their contractile forces causes the post to bend toward the block. The deflection of the post is recorded under fluorescence microscopy and analyzed using quantitative image analysis of the videos. Since a microscale post bends like a cantilever beam, its deflection can be used to quantify the forces of platelets. ResultsBlebbistatin, a myosin inhibitor, was used to confirm that deflection of the posts by the platelets in heparinized blood was due to myosin activity. When blood was incubated with 2-MeSAMP, a P2Y12 antagonist, platelets were able to aggregate, but their ability to generate contractile forces was substantially reduced. This finding indicates that ADP activation is needed for platelet contractility under shear. The rate of hemodynamic shear was found to influence platelet function, for the rate of platelet aggregation and force generation were found to increase for blood sheared from 2000 to 12,000 s-1. Moreover, platelet aggregation and contractile forces were reduced when glycoprotein Ib-V-IX complex and integrin αIIbβ3 were inhibited with antibody AK2 and antibody fragment c7E3 Fab, respectively. When citrated blood was incubated with tissue plasminogen activator, platelets aggregate and produced contractile forces that increased steadily within the first ten minutes, but then the forces began to subside. ConclusionsOur device can be used to study the role of hemodynamic shear in platelet function and gives insights into the role of platelet forces during hemostasis. Its microscale dimensions also allow us the study the biomechanics involved in the formation of a hemostatic plug during its early stages of growth and stability. [Display omitted] Disclosures:White:Vidacare Corp: Honoraria; Stasys Medical Corp: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; NIH: Research Funding; Coulter Foundation: Research Funding; Washington State Life Sciences Discovery Fund: Research Funding. Sniadecki:Stasys Medical Corporation: Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees.

Mycophenolic acid quantification in human peripheral blood mononuclear cells using liquid chromatography–tandem mass spectrometry

Background and objectivesMycophenolic acid (MPA) is an immunosuppressant widely used to prevent organ rejection after organ transplantation. MPA therapeutic drug monitoring (TDM) is currently recommended by plasma C0 – or even better – by AUC determinations, but little is known about the potential interest of measuring the intra-lymphocyte MPA concentrations, representing the target site of action. The aim of this study is to develop and validate a selective and sensitive analytical method for quantification of MPA levels in human peripheral blood mononuclear cells (PBMCs). MethodsPBMCs were extracted from heparin blood samples by Leucosep®. Methanol and MPA-d3 were used as extraction solvent and internal standard, respectively. Chromatographic separation was obtained on a XBridge BEH C18 column (2.1mm×75mm, 2.5μm) maintained at 50°C on a Waters Alliance 2795 coupled to a QuattroMicro tandem mass-spectrometer. The multiple reaction monitoring transitions used for quantification were m/z 320.97→207.0 for MPA, and 324.2→210.1 for MPA-d3, in positive ESI mode. ResultsThe total HPLC run time was 6min. The retention times for MPA-d3 and MPA were 2.55 for both compounds. The method was linear from 0.1 to 50ng/mL MPA. The coefficient r2 ranged from 0.996 to 0.998. Intra-assay and inter-assay imprecisions were <10% in the whole range of concentrations, and <20% at the LLOQ, and accuracy level was >90%. The matrix and ion suppression effects were <6%. The MPA limit of quantification was 0.1ng/mL. No interference was identified in the assay. From the preliminary results, intra-cellular MPA concentrations in kidney transplant patients ranged from 0.69 to 3.39ng/107 cells. ConclusionWe described a robust, rapid and simple method suitable for the determination of MPA concentrations in PBMCs. This method is currently used in pharmacokinetics–pharmacodynamics (PK–PD) clinical trials, in comparison to standard plasma TDM.

Analysis of cytokines and chemokines produced by whole blood, peripheral mononuclear and polymorphonuclear cells

Cytokines are immunomodulating proteins involved in cellular communication. The levels of different cytokines reflect the immune capabilities of a person. In literature both whole blood and peripheral blood mononuclear cells (PBMCs) are used, which might lead to different results. The choice between these different sources is not always explained. The goal of our experiments is to determine the cytokine response of whole blood, PBMCs and polymorphonuclear cells (PMNs) after stimulation with lipopolysaccharide (LPS). We used a multiplex analysis to determine a difference in cytokine secretion patterns. In general, PBMCs demonstrated the highest cytokine production and PMNs have an overall low cytokine production. CCL11 and interleukin-23 (IL-23) (and IL-12p40) were exclusively expressed in whole blood. IL-20, VEGF and GM-CSF were expressed only by PBMCs. This difference in expression could be explained by the bioactive components in serum, presence and interaction with granulocytes or platelets in whole blood, the anticoagulant heparin in whole blood and others. The expression of cytokines by cells is dependent on the microenvironment. Different conditions lead to different results. We recommend a thorough examination of the conditions before performing experiments.

Detecting Heparin in Whole Blood for Point of Care Anticoagulation Control During Surgery

Detecting Heparin in Whole Blood for Point of Care Anticoagulation Control During Surgery

Does protamine infusion reduce postoperative blood loss and heparin rebound in cardiac surgery patients? A retrospective analysis of 240 patients on the cardiac ICU

Cardiac surgery is associated with significant blood loss. Teoh and colleagues demonstrated a reduction in postoperative bleeding with the use of a protamine infusion and an abolishment of heparin rebound [1]. The aim of this study was to see whether the use of postoperative protamine infusions in our cardiac ITU was associated with a reduction in heparin rebound and blood loss.

Raman spectroscopy for clinical-level detection of heparin in serum by partial least-squares analysis

Heparin is the most widely used anti-coagulant for the prevention of blood clots in patients undergoing certain types of surgeries including open heart surgeries and dialysis. The precise monitoring of heparin amount in patients' blood is crucial for reducing the morbidity and mortality in surgical environments. Based upon these considerations, we have used Raman spectroscopy in conjunction with partial least squares (PLS) analysis to measure heparin concentration at clinical level which is less than 10 United States Pharmacopeia (USP) in serum. The PLS calibration model was constructed from the Raman spectra of different concentrations of heparin in serum. It showed a high coefficient of determination (R2>0.91) between the spectral data and heparin level in serum along with a low root mean square error of prediction ~4 USP/ml. It enabled the detection of extremely low concentrations of heparin in serum (~8 USP/ml) as desirable in clinical environment. The proposed optical method has the potential of being implemented as the point-of-care testing procedure during surgeries, where the interest is to rapidly monitor low concentrations of heparin in patient's blood.

Abstract TMP113: Oxidative stress and Extracellular Signal-regulated Kinase (ERK) Play a Key Role in Impairment of Receptor Desensitization and Prolongation of Vascular Reactivity in the Cerebral Artery after Subarachnoid Hemorrhage

Cerebral vasospasm is a critical determinant in the prognosis of subarachnoid hemorrhage (SAH). The vasospasm is characterized by not only enhanced but also prolonged contraction. The enhanced response is attributable to an increased receptor expression, while a prolonged response is attributable to impaired receptor desensitization. This study addressed this hypothesis and investigated the mechanism underlying a prolonged contraction, using a rabbit SAH model and A7r5 smooth muscle cells. A thrombin receptor (PAR 1 )-activating peptide (PAR 1 AP) induced a transient contraction in the basilar arteries of the control rabbits. The subsequent second stimulation produced 8% of the first response. PAR 1 AP induced an enhanced and prolonged contraction in SAH, and the second stimulation produced a 75% response. Thrombin induced an enhanced and prolonged contraction in SAH. This contraction irreversibly persisted even after terminating thrombin stimulation. The enhancement and prolongation of the contractile response after SAH was further observed with angiotensin II, vasopressin and PGF 2 α. The heparinization of autologous blood or the intrathecal application of a thrombin inhibitor, argatroban, prevented the enhancement of contraction, while they had no effect on the prolongation of the contraction. The addition of vitamin C or tempol to argatroban restored the transient response, tachyphylactic attenuation of the second response and reversibility of thrombin contraction. The amount of malondialdehyde, an indicator of oxidative stress, in the brain tissues increased after SAH, and this increase was prevented by either argatroban or vitamin C. The Ca 2+ responses to thrombin and PAR 1 AP in A7r5 cells exhibited a phenomenon similar to those seen with the contractile response in an SAH model. ERK inhibitors restored the transient response, tachyphylactic attenuation and reversibility of thrombin response, while the inhibitors of JNK, p38 kinase or Rho kinase had no effect. In conclusion, oxidative stress and ERK activation play a key role in the impaired receptor desensitization and prolonged vascular reactivity after SAH. Targeting oxidative stress and ERK could provide a novel strategy to normalize vascular reactivity after SAH.

Application of the ‘gate effect’ of a molecularly imprinted polymer grafted on an electrode for the real-time sensing of heparin in blood

Heparin is the most important anticoagulant drug used during surgeries and extracorporeal therapies. Although the blood levels of heparin should be monitored continuously during the procedure to ensure the safety of the patient, there is currently no technique for measuring heparin in real time. This study describes the use of a molecularly imprinted polymer (MIP) as a recognition element in the development of a heparin sensor for real-time monitoring. An indium tin oxide (ITO) electrode grafted with a heparin-specific MIP was used as a working electrode to perform cyclic voltammetry of ferrocyanide. The anodic current was found to be dependent on heparin concentration, probably due to the "gate effect", which is a change in the accessibility of the MIP-modified electrode to ferrocyanide, triggered by specific interaction between MIP and heparin. The kinetics of heparin interaction with the MIP-grafted electrode was evaluated using potentiostatic chronoamperometry of ferrocyanide in an electrochemical flow cell. The response time to stepwise changes in heparin concentration between 0 and 0.04 units per mL was estimated at 20 s, which is remarkably shorter than that achieved using conventional methods for monitoring heparin. The MIP-grafted electrode demonstrated exceptional sensitivity and could detect heparin in whole blood samples (0-6 units per mL) diluted 100-fold with physiological saline containing ferrocyanide. Therefore, the MIP-grafted electrode is suitable for real-time monitoring of heparin in blood. Another advantage is that a very small volume of blood is needed, which is very important, especially when regular measurements are required.

Traumatic Aortic Injury: Single-center Comparison of Open versus Endovascular Repair

BackgroundConventional open repair is a suboptimal therapy for blunt traumatic aortic injury (BTAI) due to the high postoperative mortality and morbidity rates. Recent advances in the thoracic endovascular repair technique may improve outcomes so that it becomes an attractive therapeutic option.Materials and MethodsFrom August 2003 to March 2012, 21 patients (mean age, 45.81 years) with BTAI were admitted to our institution. Of these, 18 cases (open repair in 11 patients and endovascular repair in 7 patients) were retrospectively reviewed and the early perioperative results of the two groups were compared.ResultsAlthough not statistically significant, there was a trend toward the reduction of mortality in the endovascular repair group (18.2% vs. 0%). There were no cases of paraplegia or endoleak. Statistically significant reductions in heparin dosage, blood loss, and transfusion amounts during the operations and in procedure duration were observed.ConclusionCompared with open repair, endovascular repair can be performed with favorable mortality and morbidity rates. However, relatively younger patients who have acute aortic arch angulation and a small aortic diameter may be a therapeutic challenge. Improvements in graft design, delivery sheaths, and graft durability are the cornerstone of successful endovascular repair.

Abstract 18481: Genetic Polymorphisms Associated with Response to Intraoperative Heparin Anticoagulation and Thrombosis Risk in Pediatric Patients Undergoing Cardiac Surgery

Introduction: Thrombosis is an important and widespread post-operative complication of pediatric cardiac surgery. Prophylaxis intraoperative anticoagulation in this context is obtained by using heparin which requires a bond with antithrombin to achieve anticoagulant effect. We sought to identify SNPs associated with blood antithrombin levels, response to heparin and post-operative thrombosis risk in pediatric patients undergoing cardiac surgery with cardiopulmonary bypass (CPB). Methods: A total of 625 cardiac operations with CPB in 383 patients were reviewed. Ninety-six SNPs on 53 genes in the coagulation/fibrinolysis pathways were assayed using the the Illumina GoldenGate custom SNP panel. Genotyping was successful for &gt;99% of SNPs. Regression models adjusted for repeated measures and bootstrap resampling (1,000 samples) were used to offset multiple comparisons bias; SNPs with very low minor allele frequencies were excluded. Results: Average preoperative antithrombin activity was 89±18% (normal 100%, adjusted for age and oxygen saturation) and average response to heparin was 1.0±0.3U/ml/100U/kg (blood heparin concentration achieved per 100U/kg of heparin). Post-operative thrombosis was observed after 86 operations (14%) (within 30 days of surgery, risk-adjusted according to a previously published model of post-operative thrombosis risk). SNPs associated with higher antithrombin levels, lower heparin response and higher postoperative thrombosis are listed in the Table. Conclusions: Polymorphisms in genes in the coagulation and fibrinolytic systems activity were associated with antithrombin levels, response to heparin and risk of post-operative thrombosis. The association of individual SNPs at the laboratory and clinical levels suggest a mechanism for upstream alteration in the coagulation system that is correlated with undesirable clinical outcome and could be the target for genetically tailored clinical interventions.

505 The Effect of Brand of Heparin on Peri-Operative Transfusion Rates in Patients Undergoing Cardiac Surgery

505 The Effect of Brand of Heparin on Peri-Operative Transfusion Rates in Patients Undergoing Cardiac Surgery

Phytohemagglutinin stimulation of lymphocytes improves mutation analysis of carbamoylphosphate synthetase 1

Carbamoylphosphate synthetase 1 (CPS1) is the first enzyme of the urea cycle. CPS1 deficiency is a rare autosomal-recessively inherited disorder that can lead to life-threatening hyperammonemia. Since there is no reliable biochemical marker for this disease, diagnosis relies on molecular means which is often done by RNA-based mutation analysis. Skin fibroblasts have been frequently used as a source of RNA while peripheral blood cells do not yield sufficient amounts of specific RNA. To avoid the costly and laborious use of cultured fibroblasts, we tried to use stimulated lymphocytes as an alternative. This was effectively achieved by short-term culture of full heparin blood in the presence of phytohemagglutinin. Hereby, subsequent reverse transcriptase-PCR of the CPS1 transcript became feasible and allowed to detect 16 different mutations (10 missense, 3 deletions, 2 nonsense, 1 duplication; 7 novel mutations) in 14 consecutive patients with CPS1 deficiency. When compared to retrospective data on cultured fibroblasts, the adapted method allowed substantial shortening of the median time to diagnosis (24days versus 122days, respectively). Besides disease causing mutations, we detected CPS1 transcript variants including one cryptic exon in RNA from lymphocytes with higher frequency than in RNA from fibroblasts. This underlines that all mutations found in RNA need to be confirmed by DNA sequencing.In conclusion, the presented approach improves the diagnostics of CPS1 deficiency. Besides the shortened time to diagnosis, the method is of particular importance for confirmation of findings of next generation sequencing and gene chips.

Clinical evaluation of the Sorin Xtra® autotransfusion system

The performance of the Sorin Xtra® Autotransfusion System (ATS) was studied in 62 patients undergoing coronary artery bypass grafting. Blood was collected intraoperatively and washed using three different wash sets in 4 groups. Both collected and washed blood were analysed for hemoglobin levels and hematocrit, concentrations of proteins, albumin, heparin and plasma free hemoglobin (PFH) were determined, erythrocytes, platelets and leukocytes were counted.Hematocrit measurements of the Xtra® were compared with laboratory measurements to study the accuracy of the Xtra® hematocrit sensor. In addition, the red blood cell recovery rate and elimination rates were calculated to evaluate the clinical performance of the Xtra®.The Xtra® ATS produced a volume of concentrated red blood cells with an average hematocrit from 58% to 63%, depending on the size of the bowl and the chosen default program. In all bowl sizes and programs, the Xtra® Hct-out measurement underestimated the CELL-DYN measurement by approximately 15%.The calculated recovery rates for red blood cells (RBC) in the 4 groups ranged from 86.7% to 91.6%. Elimination rates were calculated in each group for proteins (96.8-99.2%), albumin (96.4-98.7%), plasma free hemoglobin (83.6–91.2%), heparin (98.8-99.9%), platelets (82.4-94.3%) and white blood cells (28.6-42.3%).The Xtra® ATS can be appealing for its performance by producing high hematocrit levels in the washed RBC volume, while keeping RBC recovery rate at the same high level (≈ 90%) as in its predecessor, the Electa® Autotransfusion System.

Platelet Function in Stored Heparinised Autologous Blood Is Not Superior to in Patient Platelet Function during Routine Cardiopulmonary Bypass

BackgroundIn cardiac surgery, cardiopulmonary bypass (CPB) and unfractionated heparin have negative effects on blood platelet function. In acute normovolemic haemodilution autologous unfractionated heparinised blood is stored ex-vivo and retransfused at the end of the procedure to reduce (allogeneic) transfusion requirements. In this observational study we assessed whether platelet function is better preserved in ex vivo stored autologous blood compared to platelet function in the patient during CPB.Methodology/Principal FindingWe measured platelet aggregation responses pre-CPB, 5 min after the start of CPB, at the end of CPB, and after unfractionated heparin reversal, using multiple electrode aggregometry (Multiplate®) with adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP) and ristocetin activated test cells. We compared blood samples taken from the patient with samples taken from 100 ml ex-vivo stored blood, which we took to mimick blood storage during normovolemic haemodilution. Platelet function declined both in ex-vivo stored blood as well as in blood taken from the patient. At the end of CPB there were no differences in platelet aggregation responses between samples from the ex vivo stored blood and the patient.Conclusion/SignificanceEx vivo preservation of autologous blood in unfractionated heparin does not seem to be profitable to preserve platelet function.