Determination of platelet monoamine oxidase by new continuous spectrophotometric method

A simple, continuous spectrophotometric method for the determination of tissue monoamine oxidase based on the oxidation of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) using peroxidase has already been described (Ivanovi?, I. & Majki?-Singh, N. (1986) Fresenius Z. Anal. Chem. 324, 307). In the present study the method is optimised for platelet monoamine oxidase assay and applied to healthy persons and schizophrenic patients. The obtained data were statistically analysed. The continuous ABTS method is sensitive, precise (CV below 6.9%) and linear up to 83 U/g protein. Comparison with the end-point method of Szutowicz et al. (1984) Anal. Biochem. 138, 86-94) gave a good correlation (r = 0.983). The reference values for the activity of human platelet monoamine oxidase by the new continuous ABTS method are 25 to 42 U/g protein (means = 33.2 U/g protein, CV = 15.5%, n = 67). No differences were found between females and males, or between three age groups ranging from 21 to 52 years. The patients with chronic (n = 76) or acute (n = 17) schizophrenia had significantly lower monoamine oxidase activities compared with normal values (p less than 0.005), which indicates that platelet monoamine oxidase can be a possible marker for schizophrenic diseases.     

Lack of platelet activation reflected by circulating soluble glycoprotein V in pre-eclampsia

Pre-eclampsia (PE) is a human pregnancy-specific disorder of unknown aetiology. Although the quantitative relationship between platelet aggregation in PE is not clearly defined yet, we aimed to investigate the possible relationship between PE and platelet glycoprotein V (GPV), which is an integral platelet membrane protein involved in the function of the GPIb-V-IX receptor. Fifty patients with PE and 37 normotensive pregnant women (controls) were enrolled in this study. Fasting blood samples were collected and soluble GPV (sGPV) levels were determined using a commercially available enzyme immunoassay. No statistically significant difference in sGPV was found between PE patients and control subjects. There was no correlation between sGPV and platelet counts or between pregnancy duration and platelet counts. Further clinical and experimental investigations are needed to elucidate the pathological processes involved in the development of PE in complicated pregnancies.     

一种新的血小板活化释放试验监测阿司匹林的治疗反应

目的 建立一种新的血小板活化释放试验用于监测阿司匹林的治疗反应.方法 采用ARA活化抗凝全血中血小板,应用血细胞分析仪检测MPC的变化.抽取5名健康志愿者外周血标本,取血后迅速混匀,用于MPC、LTAARA和血小板CD62P表达的检测,以优化检测条件,包括ARA活化浓度(分别为0、0.5、1.0、1.5、5.0、10.0 mmol/L)的选择和ARA活化血小板时间的选择(37℃水浴中5、10、20、30、40、50、60、70、80、90 min);评价该方法的稳定性(取血后0、1、2、3 h分别检测)、重复性(MPC、LTAARA、血小板CD62P表达的检测分别重复测定10次,计算批内及批间CV);并通过体外乙酰水杨酸试验验证该方法监测阿司匹林治疗反应的可行性.ARA活化血小板后用CD61-PerCP和CD62P-PE标记,流式细胞仪测定CD62P表达阳性的血小板百分率,对MPC与CD62P的相关性进行分析.选择口服阿司匹林至少7 d的患者为服药组,共93例;选择未口服或停止口服阿司匹林7 d以上的患者为对照组,共100例.用该方法检测服药组与对照组患者ARA(终浓度0.5 mmol/L)活化前后MPC的变化,并与LTAARA方法进行比较,评价该方法的准确性.结果 ARA终浓度为0.5 mmol/L时,血小板活化充分,MPC与血小板膜表面CD62P呈负相关(r=-0.755,P<0.01).37℃水浴活化30 min后MPC达到稳定,取血后室温3 h内MPC保持稳定,新鲜全血MPC的批内CV为1.35%,固定质控全血批间CV为0.71%和0.74%.随着全血中乙酰水杨酸浓度升高(0～6.95 μmol/L),ARA活化血小板后MPC逐渐升高,CD62P逐渐减低,二者呈负相关(r＝-0.765,P<0.01).服药组MPC差值为(8.2±8.6)g/L,MPC变化率为(3.4±3.6)%,对照组分别为(37.4±10.3)g/L、(15.7±4.0)%,差异均有统计学意义(t=21.522、22.409,P均<0.01).MPC变化率ROC曲线下面积为0.992,当临界值为8.7%时,其监测阿司匹林治疗反应的敏感度为96.8%,特异度为99.0%.MPC变化率与LTAARA具有很好的相关性(r=0.720,P<0.01).结论 本研究建立了一种新的监测阿司匹林治疗反应的血小板活化释放试验,可以替代LTAARA用于临床常规检测.

Abstract：

Objective To establish a new method for monitoring aspirin response by platelet activated-release experiment.Methods The platelets in whole blood were activated by ARA,and the MPC was measured by hematology analyzer.Blood samples were drawn from five healthy volunteers for measuring MPC,LTAARA and platelet membrane CD62P expression.Blood samples were mixed thoroughly right after venipuncture. The concentration of ARA (0,0. 5,1.0,1.5,5.0 and 10. 0 mmol/L) and the time for platelet activation (5,10,20,30,40,50,60,70,80 and 90 min in 37℃ water bathe) were optimized to evaluate the stability (0,1,2 and 3 h after venipuncture) and reproducibility (MPC, LTAARA and platelet membrane CD62p were measured ten times and the CV was calculated). Platelets were mixed with acetylsalicylic acid at different concentrations in vitro in order to verify the validity for monitoring aspirin response. The percentage of CD62p positive platelets after activated by ARA was measured using flow cytometer with CD61-PerCP and CD62p-PE antibodies. The correlation between MPC and CD62P was determined. 100 patients without taking or stop taking aspirin more than 7 days and 93 patients who took aspirin at least 7 days were enrolled. Duplicate measurements of platelet function were performed using the change of MPC (ARA 0. 5 mmol/L) and LTA (ARA 0. 5 mmol/L) among two patient groups to evaluate the accuracy of the new method. Results Platelcts were completely activated by ARA at final concentration of 0. 5 mmol/L. MPC negatively correlated with platelet membrane CD62p(r=-0. 755,P<0. O1 ). MPC was stable for 30 minutes in 37 ℃ water bathe after ARA activation. The result of MPC was consistent at room temperature within 3 hours after blood collection. This method had good reproducibility. CV in batch using fresh whole blood was 1.35% and CV between batches using commercial control whole blood were 0. 71% and 0. 74%. With the concentration of acetylsalicylic acid increased (0-6. 95 μmol/L), MPC increased as CD62P decreased, which showed negative correlation (r=-0. 765 ,P <0. 01 ). The difference of MPC before and after ARA activation (ΔMPC) and MPC variance ratio of the group taking aspirin were ( 8. 2±8. 6) g/L and ( 3.4±3.6) %, and they were (37.4±10. 3 ) g/L and ( 15.7±4.0) % in control group, respectively.ΔMPC and MPC variance ratio showed significant difference between the two groups ( t=21. 522, 22. 409, all P < 0. 01 ). Area under the ROC curve for MPC variance ratio was 0. 992 with sensitivity of 96. 8% and specificity of 99.0% for monitoring the aspirin response using the cut-off of 8. 7%. MPC variance ratio had good correlation with LTAARA (r = 0. 720, P < 0. O1 ). Conclusions A new method for monitoring aspirin response by platelet activated-release experiment is established. It may replace LTAARA for routine clinical examination.     

Effects on complement activation of a new continuous autotransfusion system

Allogeneic blood transfusions may subject patients to risks of infection and allergic reactions. Various techniques for transfusion of shed blood have been developed. The aim of this study was to evaluate a new continuous autotransfusion system (Fresenius CATS^®) as regards its impact on the complement system, and on erythrocytes and leucocytes. Eighteen consecutive patients undergoing hip replacement surgery were studied. Complement variables (C4d, factor Bb, C3a and terminal complement complex, SC5b-9) and free haemoglobin, haemoglobin, leucocytes, platelets, albumin and protein were determined in the patient's blood preoperatively, 1 min before the start of transfusion, 15 and 60 min after transfusion; and in the reservoir, in the waste bag and in the retransfusion blood. Increased concentrations of C3a and SC5b-9 were found in the collected reservoir blood ( P  < 0.05). The washing and centrifugation procedure reduced these concentrations (< 0.001). High levels of free haemoglobin were found in the collected blood as well as in the processed product. The median haemoglobin level in the processed blood was 260 g L⁻¹ (range 104–289 g L⁻¹). Inflammatory mediators from the complement cascade are removed by continuous autotransfusion technique. The processed blood contains high levels of free haemoglobin.     

A new method for continuous monitoring of chest wall movement to characterize hypoxemic episodes during HFOV

Introduction  

Monitoring ventilated infants is difficult during high-frequency oscillatory ventilation (HFOV). This study tested the possible causes of hypoxemic episodes using a new method for monitoring chest wall movement during HFOV in newborn infants.     

Evaluation of a new module in the continuous monitoring of respiratory mechanics

Objective: Bedside monitoring of respiratory mechanics facilitates the use of lung protective ventilation in acute lung injury (ALI). We evaluated a new clinical monitor of respiratory mechanics.¶Design: Prospective, in vitro and in vivo study.¶Setting: University hospital.¶Patients: Measurements were done using a lung model and in patients after cardiac surgery (n = 10) and in patients with ALI (n = 10).¶Interventions and measurements: The monitor provides continuous monitoring of pressure, flow and volume waveform and loop data, and automatically collected variables of respiratory mechanics. Breath-by-breath respiratory mechanics data and the automated variables obtained with the new monitor were compared with flow and pressure reference data.¶Results: Waveform data comparison showed errors of less than 5 % for most variables. Automatically recorded respiratory pressures and volumes showed good agreement within clinical standards when compared to reference (errors from 2.5 % to 6.2 %). Automatically recorded derived variables present poor agreement (errors from 8.1 % to 158.3 %).¶Conclusions: The waveform data of the new monitor is accurate. The value of the automatically derived variables is limited by the fact that inspiratory plateau pressure and plateau compliance have no direct physiological meaning. Nevertheless, in clinical monitoring much information can be derived from the waveform signals alone and from pressure-volume and flow-volume loops. These facilitate monitoring changes in respiratory mechanics in the ALI patient.     

Evaluation of a new wireless technique for continuous electroencephalography monitoring in neurological intensive care patients

Journal of Clinical Monitoring and Computing - A novel wireless eight-channel electroencephalography (EEG) headset specially developed for ICUs was tested in regard of comparability with standard...     

A new system for continuous recurrent laryngeal nerve monitoring.

Existing nerve monitoring devices in thyroid surgery are - except for one - mainly intermittently working nerve identification tools. We present a new vagal electrode which allows true continuous monitoring of the recurrent laryngeal nerve (RLN). The electrode was designed as a tripolar hybrid cuff electrode consisting of polyimide, gold and platinum layers embedded in a flexible silicon cuff which can be opened at the long side for introducing the nerve. It is fully implantable and atraumatic. The evoked potentials are sensed by standard thyroid electrodes. Real-time signal analysis and audio feedback are achieved by specially designed software. Homogeneous and stable signals were recorded throughout the operations. Thus real-time computer-based signal analysis was possible. Evoked potentials reached 300-900 mV. Mean time to place the cuff electrode was 5.5 min. The nerve was stimulated a mean of 63 min (range 55-99 min). No RLN lesions were detected postoperatively. The new vagal electrode was easy to handle and led to stable and reproducible signals. The stimulation current could be kept extremely low due to the special geometry of the electrode. It offers the possibility for uninterrupted, continuous laryngeal nerve monitoring in thyroid surgery. In an ongoing clinical trial its compatibility as an add-on for existing nerve monitoring devices is being tested.     

新型血小板毛细管交叉配型法探讨

目的建立一种简便、易行的新型血小板交叉配型法。方法将受者血浆和供者血小板在试管中混匀并加入适量助色剂,吸入毛细管中孵育10 min。以微柱凝胶检测试验(MGT)作为对照试验方法。结果血小板配血不符时,因血小板凝集导致混合液不易从毛细管中挤出,且挤出时在生理盐水中呈蓝色条带状;血小板配型相符时,混合液能较为容易地从毛细管中挤出,且挤出后在生理盐水中迅速散开,无蓝色条带形成。检测结果与MGT一致。结论所建立血小板毛细管交叉配型法具有一定的优点,值得进一步深入研究。     

A new ultrasonic method for fluid property measurements

A new ultrasonic method for fluid property measurements is described. The method uses ultrasound to generate acoustic streaming in a fluid. The resulting flow velocity will vary due to several viscous parameters of the fluid. If there are acoustic scatterers in the fluid, it will be possible to monitor the resulting flow velocity with an ultrasound Doppler device. The same acoustic energy is then utilized both to generate the acoustic streaming in the medium and to measure the resulting flow. The method seems to be very well suited for monitoring biological processes. Of special interest, for measurements on contaminated blood or on fluids in a fermentation process, is the method's ability to investigate liquids in a completely closed vessel.     

A new method for rapid fluid bolus infusion into a peripheral vein

Objective. To compare the flow rates achieved by a new short-tubed infusion device with those obtained with a conventional apparatus, using gravity, manual pressure, and

Methods. Ten paramedic volunteers were recruited for this prospective, randomized, controlled laboratory study. For the short-tubing setup, a new device, the port, was used to adapt standard 18-cm extension tubing directly to an IV bag [short tubing/ port (STP) setup]. For the conventional (CON) setup, 280-cm tubing was used. Both study setups were tested on each of the volunteers with flow from a standard 250-mL bag of normal saline subjected to three types of driving force: 1) gravity alone, 2) pressure supplied by two hands squeezing the IV bag, and 3) a pneumatic pressure bag continuously inflated to 300 mm Hg. The mean flow rates for each driving force were compared between the two setups. Results. Using gravity flow, no significant difference was noted between the STP setup and the CON setup (0.40 vs 0.45 mL/sec, respectively, p > 0.4). However, when flow was augmented by pressure supplied by two hands squeezing the bag, mean flow was greater for the STP setup than for the CON setup (4.5 vs 2.7 mL/sec, respectively, p < 0.001). When flow was augmented by a pneumatic bag at 300 mm Hg, mean flow was again greater for the STP setup (5.6 mL/sec) than for the CON setup (3.3 mL/sec, p < 0.001).

Conclusion. Flow of crystalloid under pressure into a peripheral vein is markedly increased with the new STP setup as compared with the CON setup. Incorporation of this new setup in prehospital care would allow EMS personnel to infuse fluid more rapidly and conveniently during transport.     

Structured implicit review: a new method for monitoring nursing care quality

BACKGROUND: Nurses' independent decisions about assessment, treatment, and nursing interventions for hospitalized patients are important determinants of quality of care. Physician peer implicit review of medical records has been central to Medicare quality management and is considered the gold standard for reviewing physician care, but peer implicit review of nursing processes of care has not received similar attention. OBJECTIVE: The objective of this study was to develop and evaluate nurse structured implicit review (SIR) methods. RESEARCH DESIGN: We developed SIR instruments for rating the quality of inpatient nursing care for congestive heart failure (CHF) and cerebrovascular accident (CVA). Nurse reviewers used the SIR form to rate a nationally representative sample of randomly selected medical records for each disease from 297 acute care hospitals in 5 states (collected by the RAND-HCFA Prospective Payment System study). SUBJECTS: The study subjects were elderly Medicare inpatients with CHF (n = 291) or CVA (n = 283). MEASURES: We developed and tested scales reflecting domains of nursing process, evaluated interrater and interitem reliability, and assessed the extent to which items and scales predicted overall ratings of the quality of nursing care. RESULTS: Interrater reliability for 14 of 16 scales (CHF) or 10 of 16 scales (CVA) was > or = 0.40. Interitem reliability was > 0.80 for all but 1 scale (both diseases). Functional Assessment, Physical Assessment, and Medication Tracking ratings were the strongest predictors of overall nursing quality ratings (P < 0.001 for each). CONCLUSIONS: Nurse peer review with SIR has adequate interrater and excellent scale reliabilities and can be a valuable tool for assessing nurse performance.     

A role for glycosphingolipid-enriched microdomains in platelet glycoprotein Ib-mediated platelet activation

BACKGROUND: Glycoprotein (GP) Ib, a platelet von Willebrand factor (VWF) receptor, plays a crucial role in thrombosis and hemostasis. As recent reports have suggested that GPIb partially locates in a particular region, designated as glycosphingolipid-enriched microdomains (GEMs), we hypothesized that GEMs play a central role in GPIb-mediated platelet activation. METHODS: Platelets were stimulated by VWF/botrocetin to activate platelets through GPIb. GEMs and non-GEMs were isolated by sucrose density gradient ultracentrifugation and the location of signaling molecules characterized. The role of GEMs-mediated signaling in platelet behavior was tested by platelet aggregation and by platelet interaction with immobilized VWF under flow conditions when GEMs were disrupted by methyl-beta-cyclodextrin (MbetaCD). RESULTS: GPIb was partially translocated to GEMs upon VWF/botrocetin stimulation. Immunoprecipitation of GPIb in GEMs and non-GEMs revealed that the tyrosine kinases, Src and Lyn, were associated with GPIb only in GEMs after GPIb-stimulation, and not in non-GEMs. Activation of PLCgamma2 was more intense in GEMs than non-GEMs. Disruption of GEMs by MbetaCD strongly inhibited tyrosine phosphorylation of Syk and PLCgamma2. Functional studies revealed that stable adhesion of platelets to a VWF-coated surface under flow was impaired by GEM disruption by MbetaCD. CONCLUSION: The combined results suggest that GEMs play an important role in GPIb-mediated platelet activation.     

脉搏指数连续心排血量监测在脓毒性休克早期液体复苏中的应用

目的：评价脉搏指数连续心排血量监测（PICCO）在脓毒性休克早期液体复苏中的应用价值。方法将40例符合脓毒性休克入选标准的病例随机分为PICCO组和对照组,每组各20例,在抗感染等基本治疗基础上, PICCO组患者于PICCO监测容量指标指导下进行治疗,对照组患者参照中心静脉压指导进行常规治疗。通过对比分析两组患者复苏治疗6 h、24 h 后,血乳酸值＜2 mmoL/L 和中心静脉氧饱和度≥70％的病例数,以及24 h复苏液体量、去甲肾上腺素用量、肺水肿发生率、机械通气时间、入住ICU天数、28 d病死率等指标评价PICCO的应用价值。结果经过液体复苏6 h后,两组的血乳酸＜2 mmoL/L 和中心静脉氧饱和度≥70％的例数比较,差异均无统计学意义（χ2分别=0.11、0.11,P均＞0.05）,而经复苏24 h后两组的发生例数比较,差异均有统计学意义（χ2分别=2.75、2.98,P均＜0.05）。与对照组比较,PICCO组24 h复苏的液体量、去甲肾上腺素用量及肺水肿比率明显降低,差异均有统计学意义（t分别=-2.33、-2.27,χ2=5.16,P均＜0.05）,而两组的机械通气治疗时间、住ICU天数和28 d病死率比较,差异均无统计学意义（t分别=-1.97、-1.72,χ2=0.00,P均＞0.05）。结论 PICCO监测可以准确、可靠地评估患者容量状态,指导对脓毒性休克患者早期液体复苏治疗及血管活性药物应用,但不能明显改善脓毒性休克患者的机械通气时间、入住ICU天数、28 d病死率等方面指标。     

A Thrombelastograph whole blood assay for clinical monitoring of NSAID-insensitive transcellular platelet activation by arachidonic acid

Optical platelet aggregation (OPA) with platelet-rich plasma (PRP) was compared with a Thrombelastograph (TEG) whole blood assay for monitoring arachidonic acid (AA)-induced platelet activation. Assays were performed on 47 interventional cardiology and 24 general surgery patients receiving aspirin therapy for cardiovascular disease, as well as 48 volunteers asked to take nonsteroidal anti-inflammatory drugs (NSAIDs) or 12 volunteers on chronic NSAID therapy unrelated to diagnosed cardiovascular disease. Whole blood TEG monitoring of NSAID inhibition detected NSAID-insensitive AA activation of platelets in a significantly higher number of cardiology (23%) and surgery (25%) patients and normal volunteers on chronic NSAID (25%) therapy relative to normal subjects not on chronic NSAID therapy (0%). Whole blood NSAID insensitivity was observed with cyclooxygenase-I inhibitors, such as aspirin and ibuprofen; was not affected by Celebrex, a cyclooxygenase-II inhibitor; but was completely inhibited by thromboxane-receptor antagonists. This was not due to platelet NSAID insensitivity, because complete inhibition of AA-activation responses in PRP was observed with either TEG or OPA assays. We confirmed that thromboxane B(2) formation in PRP from NSAID-insensitive subjects was completely inhibited by NSAIDs. However, significant amounts were formed in whole blood from NSAID-insensitive subjects, but not in whole blood from NSAID-sensitive subjects. Thromboxane formation after AA addition was not found in washed blood cells with 90% reduced platelet counts or in leukocyte-rich buffy coat fractions, but could be restored by addition of PRP. NSAID-insensitive activation was inhibited by nordihydroguaiaretic acid, with an IC(50) of 30 micromol, implicating 12- and/or 15-lipoxygenases in this transcellular pathway.     

A new method for monitoring the sterility of blood donation

Sterility of blood products is a cardinal contributor to patient safety. Bacteriologic controls of stable products comply with strict regulations, but legislation imposes only limited constraints in the case of perishable products, such as packed red cells (RBCs) or fresh-frozen plasma (FFP). Therefore, it is essential to monitor the sterility of aseptic donations from uninfected donors. Such bacteriologic monitoring can now be carried out through a tertiary bag (containing a soybean casein culture medium) connected to the classical double-pack system. This system does not jeopardize the sterility of the whole system, as the connection is tightly stoppered by a membrane. After the blood drawing, this tertiary bag is filled with 5 ml of blood, and separated from the rest of the system. It is then incubated for 3 days at 30 degrees C and for 14 days at 22 degrees C, to test for eventual bacteriologic or fungal contamination. In order to check the feasibility of this technique, we studied 76 blood drawings in the control laboratory of the blood center, and the results confirm the value of this system.