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T J Greenwalt

Publications and source records attributed to T J Greenwalt.

At least 19 recordsLinked to original sources
PubMed1992

Studies in red blood cell preservation. 5. Determining the limiting concentrations of NH4Cl and Na2HPO4 needed to maintain red blood cell ATP during storage.

The purpose of the present study was to define the lowest concentrations of ammonium (NH4+) and phosphate (Pi) in an experimental additive solution (EAS) that would support suitable red blood cell (RBC) ATP levels and other in vitro characteristics for at least 84 days. It was determined that ATP maintenance was dependent upon both NH4+ and Pi concentrations. RBCs stored for 84 days in additive solutions containing 10 mM NH4+ and 0, 15, 25 and 40 mM Pi had ATP values averaging 1.87, 2.49, 2.70 and 2.65 mumol/g Hb, respectively. The shedding of exocytic hemoglobin-containing vesicles and percent hemolysis were significantly (p less than 0.001) elevated in the preservative containing 40 mM Pi. These data suggest that an EAS containing 10 mM NH4+ and 15 mM Pi would be optimal for storing RBCs up to 84 days. The extended storage would be particularly advantageous for autologous transfusion programs.

2,3-Diphosphoglycerate↗
PubMed1992

Studies in red blood cell preservation. 6. Red cell membrane remodeling during rejuvenation.

The purpose of this study was to examine whether vesiculation of RBC plays a significant role in their rejuvenation. Outdated units of Adsol blood, were divided into two aliquots and incubated with equal volumes of a solution of 100 mM pyruvate and inosine, 103 mM phosphate and 5 mM adenine (PIPA) or 0.9% saline. Following 1 h incubation, vesicles were isolated from the supernatants and quantitated for hemoglobin content. Restoration of RBC ATP, 2,3-DPG, morphology, and osmotic fragility after rejuvenation was satisfactory. The postrejuvenation mean corpuscular volumes (88.2 +/- 6.9 fl) were significantly lower (p less than 0.001) than the prerejuvenation (94.6 +/- 6.8 fl) and control (104.0 +/- 7.3 fl) volumes. The hemoglobin shed in vesicles during rejuvenation was significantly greater than in the saline controls (0.44 +/- 0.31 vs. 0.18 +/- 0.10 mg/dl RBCs; p = 0.026). These data suggest that the decreased MCV following rejuvenation is in part due to membrane loss in exocytic vesiculation.

Adenosine↗
PubMed1992

An enzyme-linked antiglobulin test to detect red cell globulins after glutaraldehyde fixation.

The purpose of this study was to develop an enzyme-linked antiglobulin test (ELAT) for IgG on RBC without the hemolysis caused by the high pH of the alkaline phosphatase reaction. This was achieved by fixing the RBC with 0.05% glutaraldehyde after attachment of the antibodies. Assays using anti-D reference standards demonstrated the sensitivity to be 1-2 ng of antibody as compared to 7.5 ng for the manual indirect antiglobulin test. The coefficients of variation of these assays ranged from 10.4 to 20.1%. The mean background absorbance at 405 nm of 105 normal RBC samples was 0.08 +/- 0.03 SD. There was an increase in sensitivity of the test after the fixed RBC were stored. Dilute glutaraldehyde stabilizes the RBC membrane and the antigen-antibody linkage resulting in a more sensitive ELAT.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

The quantification of fetomaternal hemorrhage by an enzyme-linked antibody test with glutaraldehyde fixation.

A modified enzyme-linked antiglobulin test (ELAT) for quantifying D-positive red blood cells in the circulation of D-negative pregnant women is described. The antibody-antigen bond was stabilized and the problem of hemolysis eliminated by the use of 0.05% glutaraldehyde. The r value for the standardization curves for measuring cord RBC in mixtures was 0.98; coefficient of variation 5.8%. Reproducible quantitation of 0.125% D-positive cord RBC mixed with D-negative RBC was demonstrated. In comparative studies of artificial mixtures, Kleihauer-Betke results for more than 1% cord RBC were 50-100% greater than the true values. In several examples of large fetomaternal hemorrhages, the ELAT results matched the clinical data more closely than the Kleihauer-Betke based estimates. 2 ml of fetal RBC in a 1,600-ml red cell mass can be quantified using the modified ELAT.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

An enzyme-linked antiglobulin test to quantify nanogram quantities of IgG on polystyrene microspheres.

A method is described in which polystyrene latex beads are used for constructing standard curves to estimate the number of protein molecules on cell surfaces by an enzyme-liked immunoassay test (ELAT). A series of immune globulin (IgG) dilutions in pH 9.8 carbonate buffer were coated on 3-microns microbeads by incubation overnight at 4 degrees C and subjected to ELAT. The r value of the curve derived from four assays was 0.9991. This standard curve applied to previously recorded ELAT data resulted in estimating that normal RBC have 63 +/- 19 (SD) IgG molecules and that the lower level of sensitivity of the antiglobulin test is 155 IgG molecules per RBC. The method should be useful for more precise standardization of procedures for measuring proteins on cell surfaces.

Enzyme-Linked Immunosorbent Assay↗
PubMed1991

Studies in red blood cell preservation: 4. Plasma vesicle hemoglobin exceeds free hemoglobin.

Studies were designed to find out how much of the plasma hemoglobin ( Hb) in whole blood was in microvesicles and how much was free Hb after 21 days of storage in citrate-phosphate-dextrose anticoagulant and to determine the effect of the plasticizer, di-(2-ethylhexyl)phthalate (DEHP). The total plasma Hb in polyolefin (PO) containers without DEHP was much higher than in polyvinyl chloride (PVC) with the plasticizer (p = 0.004). Less than 30% of the Hb was in free solution in either type of container. The addition of 300 micrograms/ml of DEHP to the plasma in the PO containers resulted in marked reduction in the microvesiculation (p less than 0.01) but did not affect the level of free Hb. RBC hypotonic fragility and morphology scores were significantly improved. It is concluded that microvesiculation contributes more to plasma Hb concentration than free Hb during storage. Some hemolysis of red blood cells (RBC) is expected during blood bank storage. It has been shown that part of the hemoglobin (Hb) in the suspending medium is free and part is encapsulated in microvescicles shed by the RBC [1]. The amount of hemolysis and microvesiculation that occurs has been noted to be less when blood is stored in polyvinyl chloride (PVC) containers in which di-(2-ethylhexyl)phthalate (DEHP) is used as the plasticizer [1]. The DEHP that leaches into the plasma has been shown to decrease hemolysis, microvesiculation, and the increase in osmotic fragility which RBC undergo during refrigerated storage [2-9].(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Preservation↗
PubMed1990

A method for counting leukocytes in filtered components.

Leukocyte-depleted components are being used more frequently. Quantitation of residual WRCs is needed for quality control and for establishing the critical level required to prevent alloimmunization. Electronic counting is not accurate at WBC levels below 100/microL (1) and previously proposed techniques are either tedious or require sophisticated equipment. We describe a simple hemacytometer procedure using Türks solution that gives 87-93% recovery at WBC levels below 40/microL.

Filtration↗
PubMed1990

Studies in red blood cell preservation. 1. Effect of the other formed elements.

The purpose of this study was to determine if the removal of most of the leukocytes and platelets would affect the in vitro characteristics of stored red blood cells (RBC). Fresh RBC concentrates prepared by removing platelet-rich plasma and filtration through Imugard IG 500 filters were compared with unmanipulated units after storage for up to 56 days. The filtered units were significantly better after 56 days storage for supernatant K+ (p = 0.001), hemolysis (p = 0.05), total vesicle membrane protein shed (p = 0.03), and RBC morphology score (p = 0.04). These differences occurred even though ATP levels were well maintained in both groups. The measurements that did not differ significantly were pH, hematocrit, ATP, 2,3-DPG, glucose and supernatant Na+. It is suggested that enzymes, leukotrienes, catecholamines and eicosanoids released by degenerating leukocytes and platelets may be inimical to RBC. Some may act as agonists on alpha-adrenergic and cholinergic muscarinic receptors present on RBC membranes.

Blood Platelets↗
PubMed1990

Studies in red blood cell preservation. 2. Comparison of vesicle formation, morphology, and membrane lipids during storage in AS-1 and CPDA-1.

The changes in morphology, the quantitative changes in membrane lipids and the shedding of exocytic vesicles by red blood cells (RBC) stored for 42 and 56 days in AS-1 and CPDA-1 were compared. RBC stored in AS-1 shed significantly less vesicle membrane cholesterol, phospholipid and protein and maintained better morphology scores. RBC membrane cholesterol remained higher after 56 days in AS-1 than in CPDA-1. The data suggest that during the first weeks of storage cholesterol is lost from the RBC membrane followed by a larger release of phospholipids accompanied by alterations in the phosphoinositides. The shedding of exocytic vesicles appears to be secondary to the changes in morphology resulting from the perturbation of the membrane lipids.

Adenine↗
PubMed1990

Studies in red blood cell preservation. 3. A phosphate-ammonium-adenine additive solution.

The maintenance of adenosine triphosphate (ATP) in red blood cells (RBC) during storage is largely dependent on the integrity of glycolytic metabolism in the Embden-Meyerhof pathway. Meryman et al. [Transfusion 1986; 26:500-505] hypothesized that a solution that increased the surface tension of the corpuscles through hypotonic swelling might retard the development of echinocytosis and membrane loss by the shedding of exocytic vesicles. Unexpectedly, maintenance of good ATP levels and satisfactory RBC survivals were found for a long as 18 weeks. The purpose of our study was to test their observations and to explore the possible mechanisms. Equal parts of units of packed RBC were stored in the experimental preservative and, for comparison, in ADSOL. The most notable findings were ATP values at 4 weeks averaging 5.2 mumol/g Hb (130% of initial) and at 12 weeks 2.9 mumol/g Hb (73% of initial), whereas these values declined as expected in ADSOL. Mean RBC diameters and surface areas by morphometric analysis were not significantly different in the two preservatives indicating the absence of any hypotonic swelling. The morphology scores of the RBC were significantly better throughout (p less than 0.05) than in ADSOL. The shedding of exocytic hemoglobin-containing vesicles was essentially the same in both preservatives. Our data confirm the observation that ATP levels are well maintained for at least 12 weeks, but do not show any evidence that hypotonic swelling was a part of the mechanism.

Adenine↗
PubMed1988

Effect of red cell age on vesiculation in vitro.

Microvesiculation of RBC occurs in vivo and during in vitro storage. Vesiculation of young and old populations of RBC was measured under blood bank conditions. The RBC in whole units of blood were divided by density into two approximately equal fractions in a closed system. After storage for 35 d the haemoglobin-containing vesicles were quantitatively recovered. Quantitation of protein, cholesterol and phospholipids showed significantly larger amounts in the vesicles shed by the younger RBC. Otherwise vesicles shed by the two populations of RBC did not differ.

Blood Preservation↗
PubMed1984

Erythrocyte membrane vesiculation and changes in membrane composition during storage in citrate-phosphate-dextrose-adenine-1.

Serial studies were made of the membranes of the erythrocytes and the vesicles shed during storage of blood in polyvinyl chloride containers for 35 days in citrate-phosphate-dextrose-adenine anticoagulant. Special precautions were taken to eliminate artifacts created by contaminating leukocytes, platelets and red blood cell ghosts. A total of 15.6% of the cholesterol and 5.2% of the phospholipids of the membranes was lost with no gross change in the gel electrophoretic patterns. The quantity of vesicles found in the supernatant plasma increased during storage and their membranes were characterized by the absence of spectrin, ankyrin, and periodic acid Schiff bands 2 and 3. The ratio of lipids to protein in the vesicles increased as they accumulated perhaps reflecting a rearrangement of the erythrocyte membrane constituents during prolonged maintenance at 4 degrees C.

Adenine↗
PubMed1984

Transfusion support for haemoglobinopathies.

The indications and management of blood transfusion in the haemoglobinopathies have been reviewed. The sickle cell diseases that require transfusion support are sickle cell anaemia, sickle haemoglobin-C and -D diseases and sickle beta-thalassaemia. Homozygous beta-thalassaemia (Cooley's anaemia) is the major problem among the thalassaemias. The pathophysiology of the sickle cell disorders is largely based on the secondary effects of increased blood viscosity, whereas in the thalassaemias the defect is ineffective haematopoiesis. In the former the major problems occur as manifestations of vaso-occlusive crises with disseminated bone and abdominal pain, priapism, stroke and leg ulcers. Bone infarction and aseptic necrosis occur but the widespread bone changes, underdevelopment and haemochromatosis that complicate the thalassaemia are not prominent. Transfusion therapy in the sickle cell diseases is mainly episodic and is guided by the frequency of crises and the severity of vaso-occlusive complications. Partial exchange transfusion and the maintenance of haemoglobin A concentrations at 40 to 50 per cent is frequently indicated. In the thalassaemias, maintenance of haemoglobin levels is essential for normal growth and development. The problem of haemochromatosis is very serious. With hypertransfusion regimens the haemoglobin and haemotocrit are maintained above 12-13 g/dl and 35 per cent. The resulting benefit appears to be reduced blood volume, less iron turnover, and less intestinal iron absorption. The splenomegaly in these disorders is frequently associated with hypersplenism requiring well-timed splenectomy. Chronic and intensive chelation is necessary to prevent the ravages of iron overload. The availability of automated equipment for in vivo and ex vivo blood cell separation has brought new possibilities for improving the management of these haemoglobinopathies. It is feasible, but not as yet practical, to offer transfusions of neocytes (red cells with a mean age of 30 days) which have a 50 per cent longer survival than routine red cell preparations (mean age of 60 days). Neocytes can be prepared ex vivo from fresh routine blood donations using blood cell separator devices. The result is reduced transfusion requirements. A more recent suggestion for using the new technology is to remove the patient's oldest and most abnormal corpuscles on the basis of buoyant density and replacing them with neocytes . Thus the short-lived abnormal red cells would be removed before they could unload their iron. With automation it is possible to perform these procedures on an outpatient basis.

Adult↗