Detection of intracoronary fibrin degradation after coronary balloon angioplasty.

Intracoronary thrombus formation may be involved in the pathogenesis of arterial closure after coronary angioplasty and may contribute to restenosis. It is hypothesized that, unlike markers of platelet activation and fibrin formation, D-dimer, a product of plasmin-mediated proteolysis of cross-linked fibrin, is not subject to significant catheter-induced artifact and could be used to study intracoronary fibrin degradation during angioplasty. No significant difference in D-dimer levels was noted in serial plasma samples obtained from an 8Fr arterial sheath and the wire lumen of an angioplasty balloon catheter, indicating that sampling through the catheter lumen did not induce artifactual D-dimer elevations. Translesion (proximal and distal to the lesion) coronary blood samples were collected in 31 patients undergoing elective coronary angioplasty pretreated with aspirin, dipyridamole and heparin. In 20 of those in whom translesion coronary samples for plasma D-dimer levels (mean +/- standard deviation) were collected before balloon dilation, there was no evidence of ongoing intracoronary fibrinolysis (proximal D-dimer levels, 289 +/- 145 ng/ml; distal, 299 +/- 156 ng/ml; difference not significant). After coronary angioplasty (n = 31), there was a relatively small, but significant (p less than 0.001) increase (45 +/- 71 ng/ml) in translesional D-dimer levels (proximal, 396 +/- 223 ng/ml; distal, 441 +/- 257 ng/ml). The results from this study suggest (1) D-dimer levels are not subject to significant catheter-induced artifact and may be useful for assessment of intracoronary fibrin metabolism, and (2) intracoronary degradation of fibrin can be detected after (but not before) routine coronary angioplasty despite pretreatment with antithrombotic therapy, presumably in response to balloon-induced arterial injury and fibrin formation.

Cryopreservation of dog platelets with dimethyl sulfoxide: therapeutic effectiveness of cryopreserved platelets in the treatment of thrombocytopenic dogs, and the effect of platelet storage at -80 degrees C.

Dog platelets were frozen with 6% dimethyl sulfoxide at 2-3 degrees C per minute in a -80 degrees C mechanical freezer. The frozen platelets were stored at -80 degrees C for as long as 39 months. After storage at -80 degrees C for less than 1 year, platelet in vitro freeze-thaw-wash recovery values were 70%, and in vivo survival values 1 to 2 hr after transfusion were 40% those of fresh platelets. After 2 years or longer storage, in vitro freeze-thaw-wash recovery values were 60%, and in vivo survival values 1 to 2 hr after transfusion were 20% those of fresh platelets. These results indicate that significant deterioration of the dog platelets occurred between the first and second year of storage at -80 degrees C. Platelets that were stored frozen at -80 degrees C for less than 1 year and washed before transfusion into lethally irradiated thrombocytopenic dogs were hemostatically effective.

Phosphate ion exchange resin used in the liquid preservation of baboon red cells.

Baboon whole blood, collected in 14 percent citrate-phosphate-dextrose anticoagulant solution in plastic bags was stored in 100-ml aliquots at 4 degrees C for 28 days in the presence or absence of 0.75 grams of phosphate anion exchange resin. In vitro and in vivo measurements after autologous transfusions were made to determine whether the phosphate anion exchange resin had any beneficial effect on the blood during storage. The in vitro measurements of red cell 2,3-diphosphoglycerate and P50 were higher throughout the 28 days of storage at 4 degrees C in the blood stored in the phosphate anion exchange resin. After autologous transfusions in six baboons of red cells prepared from whole blood stored at 4 degrees C for 21 days, the 24-hour posttransfusion survival values were 86 +/- 6 percent (mean +/- SD) in the presence of resin and 83 +/- 6 percent in the absence of resin. In five other baboons, red cells prepared from 28-day-old blood showed a mean 24-hour posttransfusion survival of 82 +/- 4 percent in the presence of resin and 75 +/- 4 percent in the absence of resin. The addition of a phosphate anion exchange resin to the citrate-phosphate-dextrose anticoagulant provided better maintenance of red cell 2,3-diphosphoglycerate concentrations and P50 levels during storage of whole blood at 4 degrees C, and red cells prepared from whole blood stored in this solution had better oxygen transport function than red cells prepared from blood stored without resin. Red cell adenosine triphosphate concentrations and 24-hour posttransfusion survival values were similar whether or not the anticoagulant contained resin.

Circulation and function of human platelets isolated from units of CPDA-1, CPDA-2, and CPDA-3 anticoagulated blood and frozen with DMSO.

Platelet concentrates (PC) were isolated by serial differential centrifugation from units of blood anticoagulated with one of the citrate-phosphate-dextrose-adenine solutions (CPDA-1, CPDA-2, CPDA-2). The platelet concentrates were frozen with six percent dimethylsulfoxide at 2-3 degrees C per minute and stored in a -80 degrees C mechanical freezer in polyvinyl chloride or polyolefin plastic containers. After frozen storage at -80 degrees C for up to three months, the concentrates were thawed at 42 degrees C within 2.5 to 4.0 minutes, washed with autologous plasma, two percent dimethylsulfoxide and 10 percent acid-citrate-dextrose solution, and then resuspended in plasma. The washed platelets were labeled with 51Cr and transfused back to the donor from whom they had been obtained. In vitro recovery from whole blood to platelet concentrate was 70.5 +/- 17 percent (mean +/- one SD). In vitro freeze-thaw-wash recovery determined by phase microscopy was 78.5 +/- 12.8 percent, in vivo 51Cr platelet recovery two hours after transfusion was 41.3 +/- 13.5 percent, and the platelets had a linear lifespan of about eight days. A single unit of previously frozen platelets shortened an aspirin-prolonged bleeding time two and 24 hours after infusion. Results were similar with platelets isolated from all three anticoagulants and stored in both plastics. The results also were comparable to previous findings in this laboratory with platelets isolated from ACD and CPD anticoagulated blood.

Cryopreservation of platelets isolated with the IBM 2997 blood cell separator: a rapid and simplified approach.

The equivalent of 5-7 units of platelets, isolated from a single donor with the IBM Blood Cell Processor 2997 using a dual stage separation chamber, was frozen with the cryoprotectant dimethylsulfoxide (DMSO). The DMSO-saline solution was added directly to the platelets, and the platelets were frozen in a polyvinyl chloride plastic bag by storage in a -80 degrees C mechanical freezer. Washing the thawed platelets with a phosphate-buffered sodium chloride-dextrose solution, pH of 5.0, removed about 95% of the DMSO. In vitro freeze-thaw-wash recovery was 80%, and in vivo 51Cr platelet recovery was 31%. Platelet dense body granules were well maintained after freezing, thawing, and washing. This is a safe and effective method of platelet cryopreservation which can be performed in less time than other currently used methods.

Enumeration of previously frozen platelets using the Coulter Counter, phase microscopy, and the technicon optical system.

The Coulter Counter, phase microscopy, and the Technicon optical system were used to enumerate platelets in samples of whole blood, platelet-rich plasma, platelet concentrates before and after addition of DMSO, and on platelet concentrates that had been frozen, thawed, and washed. We observed agreement among the three counting methods when platelet counts were determined in whole blood, platelet-rich plasma, and platelet concentrate before and after DMSO addition. Enumeration after the cryopreservation process, however, showed highly significant differences among the counting systems. Platelet counts on platelet concentrates after freezing the thawing performed with the Coulter Counter were 25 per cent greater than with phase microscopy and 55 per cent greater than with Technicon. Counts with phase microscopy were 30 per cent greater than Technicon values. These data indicate that the method used to enumerate previously frozen platelets affects the apparent platelet count.

Cryopreserved red blood cells for pediatric transfusion. Frozen storage of small aliquots in polyvinyl chloride (PVC) plastic bags.

Human nonrejuvenated and rejuvenated red bood cells were prepared for cryopreservation and subsequent pediatric transfusion. Glycerol was added to the red blood cells in the primary polyvinyl chloride plastic collection bag to achieve a concentration of 40 per cent W/V. The red blood cells were concentrated by centrifugation, and the supernatant glycerol was discarded. Each glycerolized unit was divided into four equal aliquots in the individual 600-ml bags of a dry quadruple polyvinyl chloride plastic system, and each aliquot was frozen and stored at -80 C. After thawing, sodium chloride solutions were used to wash the aliquots in the IBM Blood Processor 2991-1 or 2991-2 or the Haemonetics Blood Processor 115, and the washed aliquots were stored in a sodium chloride-glucose-phosphate solution at 4 C for 24 hours. Freeze-thaw recovery of the red blood cells was about 97 per cent, and freeze-thaw-wash recovery was about 84 per cent. Twenty-four-hour posttransfusion survival values were about 92 per cent for both nonrejuvenated and indated-rejuvenated red blood cells. Nonrejuvenated red blood cells, those frozen within three to five days of collection without biochemical modification, had normal oxygen transport function at the time of transfusion; rejuvenated red blood cells, those biochemically treated with PIGPA Solution A after three to five days of storage at 4 C, had improved oxygen transport function at the time of transfusion.

Studies on the in vivo elution of 51Cr from baboon red blood cells.

Four anticoagulant solutions were added to baboon red blood cells prior to labeling with 51Cr to determine how each would influence the distribution of 51Cr within the red blood cells, the loss of 51Cr from the red blood cells after transfusion, and the calculated red cell survival value. The 51Cr label was detected in the hemoglobin and in the low molecular weight compounds within the red blood cells. The elution of 51Cr from labeled baboon red blood cells following transfusion could not be explained by the distribution of 51Cr between hemoglobin and low molecular weight compounds within the red blood cells.

Freezing in the primary polyvinylchloride plastic collection bag: a new system for preparing and freezing nonrejuvenated and rejuvenated red blood cells.

Red blood cells were stored at 4 C in the primary bag with an integrally attached empty transfer pack so that the red blood cells could be rejuvenated or not, as desired before glycerolization and freezing. The rejuvenation and glycerol solutions were added through ports in the system. After glycerolization, the red blood cells were concentrated by centrifugation to remove the supernatant glycerol before freezing with 40% w/v glycerol in the primary polyvinylchloride (PVC) plastic container at -80 C. After thawing, the red blood cells were washed using either the Haemonetics Blood Processor 115 or the IBM Blood Processor 2991-1 or 2991-2. In each system, 50 ml of 12% sodium chloride and 1.5 to 1.6 liters of 0.9% sodium chloride-0.2% glucose-25 meq/l disodium phosphate were used. Recovery of red blood cells in vitro was 91 per cent. After three days of postwash storage at 4 C, nonrejuvenated red blood cells had a mean 24-hour posttransfusion survival of 88 per cent, and outdated-rejuvenated red blood cells a value of 81 per cent. This new system is simpler and safer than methods previously used in this laboratory, and red blood cell recovery and 24-hour posttransfusion survivals were comparable or better.

Improved oxygen delivery to the myocardium during hypothermia by perfusion with 2,3 DPG-enriched red blood cells.

The oxygen affinity of red cells increases stepwise with temperature reductions below 37 degrees C. In vitro studies demonstrated that biochemically modified red cells with increased 2,3 diphosphoglycerate (2,3 DPG) (150% and 250% of normal) exhibited significantly less oxygen affinity at 24 degrees C than did unmodified cells. At 15 degrees C, significant attenuation of affinity was observed with 250%, but not 150%, of normal 2,3 DPG cells. Measurements made of isolated fibrillating dog hearts during perfusion at 24 degrees C alternately with unmodified (80% of normal 2,3 DPG) and modified (300% of normal 2,3 DPG) red cells demonstrated significantly greater oxygen consumption, higher coronary sinus partial pressures of oxygen and carbon dioxide, higher in vitro P50 values, and lower arterial and coronary sinus lactate levels during perfusion with modified as compared with unmodified cells. This evidence, indicating improved oxygen delivery to hypothermic dog hearts by red cells with 300% of normal 2,3 DPG activity, suggests that high 2,3 DPG cells might protect myocardial tissue in patients undergoing hypothermic cardiac operation.

Cryopreservation of human platelets isolated by discontinuous-flow centrifugation using the Haemonetics Model 30 Blood Processor.

Platelets were isolated from normal volunteers by discontinuous-flow centrifugation using the Haemonetics Model 30 Blood Processor. The numerical equivalent of about five single units of platelets collected at each pheresis were frozen together in a -80 C mechanical freezer with a 6% final concentration of dimethylsulfoxide (DMSO) as the cryoprotectant. Platelet freeze-thaw-wash recovery in vitro was about 80 per cent and the platelet recovery value depended upon the method used to enumerate the platelets. The 51Cr survival values in vivo were about 50 per cent less than those in fresh platelets. These values were not significantly different from those seen when platelets were isolated from single units of blood by differential serial centrifugation. Transfusion of two and one-half units of freeze-preserved platelets provided an increase in the recipient's circulating platelet count comparable with that from one unit of fresh platelets. The hemostatic effectiveness of freeze-preserved platelets isolated by discontinuous-flow centrifugation has not yet been studied.

Nuclear magnetic resonance studies of blood platelets.

Blood platelets contain membrane-enclosed granules which have inside them high concentrations of 5-hydroxytryptamine (serotonin) along with adenine nucleotides and divalent metal ions. 19F n.m.r. of fluorinated serotonin incorporated into the granules of both human and pig intact platelets has shown that the motional state of the serotonin is restricted. Comparison with 31P n.m.r. experiments indicates that this restriction of motion is a consequence of high molecular weight aggregates formed by the adenine nucleotides and metal ions, and that it varies with the species from which the platelets are obtained. In the case of human platelet granules, at least, these high molecular weight aggregates are present in the absence as well as in the presence of serotonin. The biological significance of these data is briefly discussed.

Therapeutic effectiveness and safety of outdated human red blood cells rejuvenated to improve oxygen transport function, frozen for about 1.5 years at 80 C, washed, and stored at 4 C for 24 hours prior to rapid infusion.

After storage at 4 C for 20 to 28 days, red blood cells were biochemically modified to improve their oxygen transport function which had deteriorated during liquid storage. The solution used for rejuvenation contained pyruvate, inosine, glucose, phosphate, and adenine (PIGPA Solution B). The rejuvenated red blood cells were frozen with 40% W/V glycerol in a polyolefin plastic bag and were stored in the frozen state for about 1.5 years at -80 C. After thawing and washing the red blood cells were stored at 4 C in a sodium chloride-glucose-phosphate solution for 24 hours before transfusion. A pool of four to ten units was rapidly transfused to each of 14 elderly anemic recipients, 11 of whom had cardiopulmonary insufficiency. Recovery of the red blood cells after the freeze-thaw process was about 97 per cent, and after the freeze-thaw-wash process about 90 per cent. The 24-hour posttransfusion survival values were about 75 per cent, and the long-term survival values were about 85 days depending on the disease state of the recipient. The red blood cells had 1.5 times normal 2.3-DPG levels and a decreased affinity for oxygen at the time of transfusion and were able to delivery oxygen at high oxygen tension immediately after the rapid infusion of pools of from four to ten units through a 40-or 170-micron filter. Plasma hemoglobin levels were consistent with extravascular sequestration of nonviable red blood cells, and uric acid levels were not increased during the immediate 24-hour posttransfusion period.

A primate model for the study of the interaction of 111In-labeled baboon platelets with Dacron arterial prostheses.

This paper presents early experience with a primate model for the noninvasive study of the interaction of circulating platelets with healing arterial prostheses. These experiments demonstrate that baboon platelets can be isolated and labeled with 111Indium with high efficiency using a sterile technique. Platelets subjected to this process have a linear life span similar to that of 51Chromium-labeled baboon platelets. The high energy gamma emission of 111Indium oxine allows for external scanning using a standard gamma camera. The small quantity of 111Indium-labeled platelets in the region of the graft can be discriminated from the surrounding blood vessel and quantitated by gamma camera imaging and computer analysis. There was a significant increase in the platelet deposition on prosthetic surfaces observed 5--48 hours after graft implantation and injection of 111Indium-labeled autologous platelets.