Clogging-free continuous operation with whole blood in a radial pillar device (RAPID)

Pillar-based passive microfluidic devices combine the advantages of simple designs, low device footprint, and high selectivity for size-based separation of blood cells. Most of these device designs have been validated with dilute blood samples. Handling whole blood in pillar-based devices is extremely challenging due to clogging. The high proportion of cells (particularly red blood cells) in blood, the varying sizes and stiffness of the different blood cells, and the tendency of the cells to aggregate lead to clogging of the pillars within a short period. We recently reported a radial pillar device (RAPID) design for contin-uous and high throughput separation of multi-sized rigid polystyrene particles in a single experiment. In this manuscript, we have given detailed guidelines to modify the design of RAPID for any application with deformable objects (e.g. cells). We have adapted RAPID to work with blood samples directly without any pre-processing steps. We were successful in operating the device with whole blood for almost 6 hours, which is difficult to achieve with most pillar-based devices. Finally, we demonstrated up to ~ 60-fold enrichment of platelets as an illustration of the improved device design. Whole blood pillar-based platelet clog-free RAPID

All-Blood (Miniplegia) Versus Dilute Cardioplegia in Experimental Surgical Revascularization of Evolving Infarction

Background The advantages of blood cardioplegia include the oxygen-carrying capacity, superior oncotic and buffering properties, and endogenous antioxidants contained in blood. However, the partial dilution of blood in 4:1 (blood:crystalloid) cardioplegic solutions may nullify these advantages and progressively dilute blood during continuous retrograde delivery. This study tested the hypothesis that all-blood (66:1) cardioplegia provides superior myocardial protection compared with dilute (4:1) cardioplegia delivered in a continuous retrograde modality during surgical reperfusion of evolving myocardial infarction. Methods and Results After 60 minutes of left anterior descending coronary artery (LAD) occlusion, anesthetized canines were placed on cardiopulmonary bypass and randomized to either all-blood cardioplegia (AB group) or dilute blood cardioplegia (Dil group). After cross clamping, arrest was induced with 5 minutes of tepid (30°C) antegrade potassium all-blood or dilute blood cardioplegia and maintained with tepid retrograde coronary sinus cardioplegia for a total of 1 hour. The LAD was released after 30 minutes of arrest, simulating revascularization. The cardioplegia hematocrit for the Dil group was lower than that for the AB group (7±1% versus 12±2%, P <0.05); at the end of bypass, systemic hematocrit was lower in the Dil group than in the Ab group (15±1% versus 20±1%, P <0.05). Infarct size (triphenyltetrazolium chloride staining) was comparable between the AB and Dil groups (29.6±2.9% versus 30.3±3.9% of area at risk), and there was no difference in area-at-risk myocardium systolic shortening (by sonomicrometry, −0.3±1% versus −0.4±1%). Tissue edema after bypass tended to be greater in the Dil group compared with the AB group in the heart (82±0% versus 81±1%), lung (79±1% versus 78±1%), liver (75±1% versus 74±0%), and skeletal muscle (76±1% versus 73±2%) and was significantly greater in the duodenum (80±1% versus 79±1%, P <0.05) and kidney (82±1% versus 79±1%, P <0.05). Postexperimental endothelial function (relaxation of acetylcholine) was impaired in LADs of the AB group versus the Dil group (59±6% versus 77±5%, P <0.05). Conclusions Both all-blood cardioplegia and dilute cardioplegia have disadvantages, but these do not have an impact on the pathogenesis of infarct size or recovery of regional contractile function.

α2-Antiplasmin, Plasminogen Activator Inhibitor (PAI) and Dilute Blood Clot Lysis Time in Selected Disease States

SummaryThis paper is an attempt to assess the relevance of the inhibitors of fibrinolysis for clot lysis in selected disease states and to discuss the mechanisms leading to acquired abnormal levels of such inhibitors. When compared to 20 control subjects the 30 hypertriglyceridemic patients (14 with type IIb and 16 with type IV) displayed significantly (p <0.001) increased plasma plasminogen activator inhibitor (PAI) activity (221 ± 88% and 290 ± 104% respectively; mean ± SD), moderately (p <0.01) increased α2 antiplasmin (α2AP) level (112 ± 11% and 115 ± 16%) and accordingly an obviously prolonged dilute blood clot lysis time (DBCLT). Neither PAI activity and α2AP level nor DBCLT were significantly different from controls in the 10 patients with hyperlipoproteinemia type IIa. The 18 patients with severe hepatic cirrhosis had low α2AP level (59 ± 19.7%) and accelerated clot lysis, while mean PAI activity (160 ± 87%) was slightly (p <0.05) increased. In the 17 nephrotic patients α2AP was increased (115 ±12%) while PAI activity was similar to controls and DBCLT rather shorter. Two liver secretion enzymes, namely serum Cholinesterase and plasma protein C, were found to be decreased in cirrhotic patients, similar to control values in hyperlipoproteinemia type Ha and obviously increased in nephrotic patients as well as in hypertriglyceridemic subjects. The relevance of PAI and α2AP for clot lysis was considered in relation to data in the literature concerning the behaviour of t-PA and factor XIII. Enhanced hepatic synthesis of protease inhibitors and factor XIII as a possible cause of delayed clot lysis in hypertriglyceridemia was envisaged.

Tissue-Type Plasminogen Activator (t-PA) and Dilute Blood Clot Lysis Time in Nephrotic Patients

SummaryWhen compared to normal weight normolipidemic control subjects, dilute blood clot lysis time was found to be obviously (p <0.001) prolonged in hypertriglyceridemic patients without proteinuria and slightly (p <0.05) accelerated in hyperlipidemic nephrotic patients in spite of their very high levels of plasma fibrinogen. As a result the ratio plasma fibrinogen (mg/dl) per clot lysis time (minutes) was 1.241 ± 0.08 (X ± SEM) in control subjects, 0.574 ± 0.07 in hypertriglyceridemic patients and 2.69 ± 0.172 in nephrotic patients. This finding suggesting that a larger amount of fibrin is rather readily dispersed from dilute blood clots of nephrotic patients was associated with higher levels of plasma t-PA: Ag (9.45 ng/ml ± 1.18 in nephrotic patients versus 5.8 ng/ml ± 1.23 in controls before venous occlusion and respectively 33.1 ng/ml ± 3.83 versus 20.3 ± 3.40 in controls after venous occlusion). Plasminogen activator activity of the euglobulins as assessed by the bovine fibrin-agarose plate was significantly higher in nephrotic patients only after venous occlusion. Plasma samples of nephrotic patients exerted a more potent inhibition of fibrinolysis in a urokinase activated system. This effect was, however, mainly due to the high levels of α2 macroglobulin in nephrotic plasma which apparently have little influence on dilute blood clot lysis time.

The Factor XII-Independent Plasminogen Proactivator System Of Plasma Includes Urokinase-Related Activity

Antibodies to urokinase have been shown to quench part of the fibrinolytic activity of plasma, not involving the extrinsic system. Previously, the intrinsic system has been subdivided into two parts, one dependent and another independent of factor XII, for activation. Both represent approximately 50 BAU activator activity/ml plasma. The aim of this study was to further locate the UK-related activity within the intrinsic fibrinolytic pathways and to delineate its participation in fibrinolysis tests in vitro.Antibodies to UK (AUK) were shown not to quench activities of contact factors such as factor XII, prekallikrein and factor XI in coagulation and chromogenic substrate assays. AUK inhibited in normal plasma, Hageman trait and Fletcher trait plasma a discrete portion of approximately 50 BAU activator activity/ml, apparently the activator activity derived from the factor XII-independent system.Immunoadsorption of plasma with immobilized AUK resulted in a depletion of approximately 50 BAU activator activity/- ml. The contact factors were undisturbed, as were plasminogen and the extrinsic activator. Additions of UK did not restore lost activity, suggesting that AUK has removed the proactivator component.The amount of UK-related activator activity compares to approximately 0.4 IU urokinase activity/ml, and shows a fairly constant level in individuals and is not enhanced by known stimuli for extrinsic activator release.Significant contributions to blood fibrinolytic activity in current assay systems, including the dilute blood clot lysis time method could be demonstrated.

In Vitro Effect of p-Chlormercuribenzoate upon Dilute Blood Clot Lysis Time in Hyperlipemia

SummaryIn comparison to values obtained in normal-weight normolipenic controls, dilute blood clot lysis time was found to be prolonged in resting and exercised patients with endogenous hypertriglyceridemia. Lysis time was also prolonged in overweight subjects with only slightly increased serum lipids but it was not significantly changed in type II-a hyperlipoproteinemia. An obvious acceleration of clot lysis was noted in patients with decompensated cirrhosis of the liver. Inclusion of p-chlormercuribenzoate (PCMB) into the diluted blood clot caused an acceleration of lysis time. This effect was particularly obvious with hypertriglyceridemic blood and minimal with blood obtained from cirrhotic patients. Addition of various amounts of heat-defibrinated plasma to blood from cirrhotic patients caused a proportional prolongation of clot lysis time and this effect was greatly diminished in the presence of PCMB. Similar amounts of serum had a much lesser effect than defibrinated plasma. Inhibition of fibrin cross-linking by PCMB was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis.The present data complete previous observations concerning a higher plasma factor XIII activity in type II-b and type IV hyperlipoproteinemia and suggest that an increased crosslinking of fibrin might partially explain the deficient thrombolysis of hypertriglyceridemic patients.