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M D Klein

Publications and source records attributed to M D Klein.

174 records · Page 10Linked to original sources
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Sensitivity of the Azure heparin assay and comparisons with activated clotting time and whole blood partial thromboplastin time.

In order to compare the Azure A Assay to other commonly accepted measures of heparinization, samples of fresh donor sheep blood were prepared with known levels of heparin between 0 and 9 U/ml. For each sample, two determinations of the Activated Clotting Time (ACT) and Whole Blood Partial Thromboplastin Time (WBPTT) were performed. Similarly, an Azure A Assay standard curve of absorbance versus heparin concentration was constructed for the same blood. Three replicates of the procedure were performed, and the average relative error in each assay was computed as the 95% confidence interval on the regression line divided by the slope of the regression curve. Parallel studies were also performed to assess the effect of various plasma constituents on the Azure Assay. Results indicate that all of the assays are equally accurate for heparin levels at or below 2 U/ml, while the ACT and Azure assays are equivalent at heparin levels near 4 U/ml, but the WBPTT is not usable at concentrations above 2 U/ml. The Azure Assay is not sensitive enough for very low heparin levels (less than 1 U/ml), but it is quite accurate and, indeed, is the only rapid assay for heparin concentrations higher than 4 U/ml. In the sensitivity studies, variations in albumin levels and plasma platelet count were shown to have a significant effect on the accuracy of the Azure assay, whereas calcium levels had no effect.

Animals↗
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Is heparin necessary for extracorporeal circulation?

To explore the possibility of extracorporeal circulation (ECC) without heparin, we performed veno-arterial extracorporeal membrane oxygenator procedures for a five hour period on a total of 15 rabbits in three groups. Blood flow rates were maintained at 125 ml/min with a standard roller pump through 1/4 inch PVC tubing and a 0.4 m2 SciMed membrane oxygenator. The rabbits in Group 1 received a standard dose of heparin, while no form of anticoagulation was used in Group 2. The tubing and membrane surfaces in Group 3 were modified by a glutaraldehyde crosslinked albumin-heparin complex with no systemic heparinization. Blood samples were obtained for activated clotting time, platelet count, and blood gas determinations. Circuits were evaluated for thrombosis, and samples of lung and kidney were examined histologically for emboli. There were no statistically significant differences among the three groups for any of the quantitative parameters. Moderate thrombus formation was visually detected in some areas of stagnation in the nonheparinized systems, while the albumin coating appeared to inhibit thrombus formation in regions of moderate and high blood flow. These results do not reflect any significant benefit of either the albumin-heparin coating or systemic heparinization during short-term ECC.

Albumins↗
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Quantitative scanning electron microscopy for the evaluation of thrombosis in extracorporeal circuits.

Methods devised in the past to evaluate blood-material interactions in extracorporeal devices assess small segments of test materials and do not analyze the entire circuit. In order to develop an effective method for the control of thrombosis in extracorporeal circulation, it is important to perform a quantitative evaluation of random samples of all the circuit elements. In an effort to quantify thrombosis in experimental extracorporeal membrane oxygenation circuits, random samples of silicone rubber membrane and polyvinyl chloride tubing were examined using scanning electron microscopy (SEM) and quantitative morphometry. The results indicate no statistically significant differences in the amounts of various adhered blood components between the tubing and the membrane. Greater than 60% of the examined surface had no blood components present. Platelet adhesion on the surface was predominant. Evidence of fibrin, gross thrombi, red cells, and white cells in small quantities were noted. It is concluded that quantitative scanning electron microscopy (QSEM) not only provides accurate information about the morphologic changes of blood cells due to surface contact, but also quantifies the individual elements and therefore assesses their contributions to thrombosis. This quantitative description will allow more meaningful comparison of blood-compatible materials and surfaces.

Animals↗
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Prolonged extracorporeal membrane oxygenation in sheep with a hollow-fiber oxygenator and a centrifugal pump.

ECMO with a roller-pump employs two potentially thrombogenic devices: the servoregulator bladder and heat exchanger. To eliminate these we used an 0.8 m2 Capiox II hollow-fiber oxygenator ventilated with warmed humidified oxygen and a Bio-Medicus centrifugal pump with a 1/4 inch head at a flow of 250 cc/min in six sheep during 96 hr of ECMO each. Oxygenator performance and plasma Hgb were determined, as were the volume, electrolyte, and protein content of fluid accumulating in the gas phase of the oxygenator. Mean oxygen transfer was 15.78 +/- 3.15 ml/min, and mean differences in PaO2 between blood entering and leaving the oxygenator was 360 +/- 49 mmHg. Mean plasma Hgb was 24.7 +/- 12.8 mg/dl. Fluid in the gas phase of the oxygenator was less than 75 ml/day and contained no albumin, protein, sodium, potassium, or chloride. Centrifugal pumps have been associated with hemolysis at low flows, but this may have been due to the simultaneous use of silastic membrane oxygenators that have high resistance. Hollow-fiber oxygenators have been associated with early failure of gas exchange and fluid accumulation in the gas phase. This may have been due to ventilation with dry gas. We conclude that a hollow-fiber oxygenator and a centrifugal pump can provide excellent gas exchange, acceptable hemolysis, and little fluid loss.

Animals↗
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Neonatal ECMO.

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Cerebral Hemorrhage↗
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Feasibility of a photosynthetic artificial lung.

The success of extracorporeal membrane oxygenation (ECMO) for the treatment of acute respiratory failure has led to consideration of the development of a more portable, and perhaps even implantable, artificial lung. The authors suggest a bioregenerative life support system that includes a photo-synthetic organism that can remove CO2 and produce O2 in the presence of an energy source. To build a model of such a photosynthetic artificial lung, the photosynthetic capability of a high temperature strain of the algae Chlorella pyrenoidosa was maximized at a cell density of 25 million cells/ml to serve as the O2 producer and CO2 remover. The "patient" in this model was comprised of 1 L of medium or 350 ml of blood, interfaced with the photosynthetic system across a gas transfer membrane. The experiments demonstrated the ability of the plant cells to supply O2 and remove CO2 from the "patient" with a maximum rate of 0.55 mmoles/L/hr under the most favorable measured operating conditions. The projected rate of 1.0 mmoles/L/hr required for physiologic applications is not totally ab absurd idea, with a slightly modified set-up. Modifications may be in the form of regulating the photosynthetic pathway or genetically engineering a hybrid strain with enhanced O2 producing and suppressed photoinhibition capacity.

Artificial Organs↗
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Rapid hepatocyte spheroid formation: optimization and long-term function in perfused microcapsules.

Enhancement of cell-cell interactions and, hence, long-term function in liver support systems can be effected by controlling the diameters of hepatocyte aggregates or spheroids. In this study, primary rat hepatocytes were induced to rapidly form spheroids using an intermittent settling and agitation protocol. The cells were seeded into albumin coated flasks at densities ranging from 80,000 to 520,000 cells/cm2. Hepatocytes were resuspended for 15 sec at 20-min intervals by placing the flasks on a timer controlled linear shaker. At time points ranging from 8 to 24 hr, hepatocyte aggregates were imaged via light microscopy. Mean spheroid diameter and shape factor were determined using computer analysis of captured images. Spheroid diameter could be controlled within the range of 60 to 240 microns. For long-term evaluation, spheroids were microencapsulated and cultured for 21 days under perfusion conditions. Encapsulated spheroids secreted albumin at rates comparable to collagen sandwich control cultures for at least 14 days, with peak rates (approximately 80 microns/day/10(6) cells) exhibited after culture medium changes. The results show that controlled, high efficiency hepatocyte aggregation can be accomplished in as little as 8 hr, and that the encapsulated spheroids exhibit long-term in vitro function.

Albumins↗