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A novel method developed in this laboratory [D.J. Moore et al., Biochemistry 35 (1996) 229-235; D.J. Moore et al., Biochemistry 36 (1997) 660-664] to study the conformational order and the propensity for domain formation of specific phospholipids in intact human erythrocytes is extended to two additional species. Acyl chain perdeuterated 1,2-dilauroylphosphatidylethanolamine (diC12PE-d46) was incorporated preferentially (in separate experiments) into the inner leaflet of stomatocytic erythrocytes and into the outer leaflet of echinocytic erythrocytes, while acyl chain perdeuterated 1,2-dipentadecanoylphosphatidylcholine (diC15PC-d58) was incorporated into the outer leaflet of echinocytic erythrocytes. The conformational order and phase behavior of the incorporated molecules were monitored through FT-IR studies of the temperature dependence of the CD2 stretching vibrations. For both diC12PE-d46 and diC15PC-d58, the gel-->liquid crystal phase transition persisted when these lipids were located in the outer leaflet of echinocytic cells, a result indicative of the persistence of phospholipid domains. In each case, the transition widths were broadened compared to the pure lipids, suggestive of either small domains or the presence of additional molecular components within the domains. The conformational order of diC12PE-d46 differed markedly depending on its location and the morphology of the cells. When located predominantly in the inner membrane of stomatocytes, the phase transition of this species was abolished and the conformational order compared with pure lipid vesicles at the same temperature was much lower. The current results along with our previous studies provide a sufficient experimental basis to deduce some general principles of phospholipid conformational order and organization in both normal and shape-altered erythrocytes.

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Acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) and dimyristoyphosphatidylserine (DMPS-d54) were incorporated into human erythrocytes. Light microscopy demonstrated that erythrocytes incubated with an equimolar mixture of DMPC-d54/DMPS or DMPC/DMPS-d54 remained mostly discocytic whereas cells incubated with either DMPC-d54 or DMPS-d54 alone became echinocytic or stomatocytic, respectively. Cells in which the aminophospholipid translocating protein was inhibited became echinocytic when incubated with DMPS-d54. Fourier transform infrared (FTIR) spectroscopy was used to monitor conformational order in the acyl chains of the incorporated phospholipid, as detected through the asymmetric CD2 stretching vibrations in the intact cells. In cells incubated with equimolar mixtures of DMPC-d54/DMPS or DMPC/DMPS-d54, the deuterated species exhibited no thermotropic phase transitions but revealed chain order intermediate between the gel and liquid-crystal states. In contrast, DMPS-d54 incorporated into the outer leaflet of echinocytic erythrocytes was conformationally ordered while the same species incorporated into the inner leaflet of stomatocytic erythrocytes was highly disordered at all temperatures studied. Finally, DMPC-d54 incorporated into the outer leaflet of echinocytic erythrocytes exhibited a phase transition, suggesting that this species persists in domains. These data indicate that the acyl chain conformational order of specific phospholipids in the intact human erythrocyte is changed with alterations in cell morphology.

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Acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) and dimyristoylphosphatidylserine (DMPS-d54) were incorporated by incubation into human erythrocytes. Light microscopic analysis demonstrated that erythrocytes incubated with DMPC-d54 became echinocytic while those incubated with DMPS-d54 became stomatocytic. This indicates that DMPC-d54 was incorporated preferentially into the outer monolayer whereas DMPS-d54 was selectively incorporated into the inner monolayer. Fourier transform infrared (FTIR) spectroscopy was used to monitor the conformational order of the incorporated phospholipids. The asymmetric CD2 stretching frequency of the inserted perdeuterated acyl chains was measured in both isolated membranes and intact erythrocytes as a function of temperature. DMPC-d54 incorporated into erythrocytes exhibited a cooperative phase transition at approximately 19 degrees C, i.e., at the same temperature as pure vesicles. In contrast, DMPS-d54 incorporated into red cells exhibited no phase transition, but possessed conformational order similar to that of the liquid-crystalline state. These results suggest that DMPC-d54 persists in domains in the outer monolayer while DMPS-d54 is dispersed in the inner monolayer. These experiments are the first to demonstrate that FTIR spectroscopy can be utilized to monitor directly a specific species of lipid molecule from the entire phospholipid population.

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The indications for bone marrow examination in children are varied and complex. In children who have isolated anemia, marrow examination usually is limited to those who have severe or persistent normocytic anemia in the absence of blood loss or hemolysis. Bone marrow studies are not necessary in children who have reactive lymphocytes; in most children who have leukopenia, leukocytosis, or thrombocytosis; and in many who have ITP. Marrow examination always is indicated when leukemia is suspected, in children who have pancytopenia of unknown etiology, and when metastatic involvement of the marrow is suspected. If one believes that examination of the bone marrow may be indicated, consultation with a pediatric hematologist/oncologist is recommended. The consultant can verify the need for the examination, decide what specific marrow studies should be performed, and suggest what other laboratory studies could be helpful.

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We used Fourier transform infrared spectroscopy (FT-IR) to quantitatively monitor peroxidative damage in model phospholipids and in human erythrocyte membranes. The 3012-cm-1 band arising from olefinic C-H stretching modes diminished in intensity during membrane peroxidation as a result of loss of unsaturation in the phospholipid acyl chains. A precise (correlation coefficient = 0.99) linear relationship between the number of C = C bonds in the chains and the fraction of the total C-H stretching contour occupied by the olefinic C-H stretch was established for model phospholipids. Intact human erythrocytes and isolated ("ghost") membranes were also peroxidized and the loss of C = C bonds was demonstrated by FT-IR. GC studies of erythrocyte membranes confirmed that the primary site of peroxidation occurred at phospholipids containing arachidonate acyl chains. Finally, the formation of a new feature at 1260 cm-1 in the FT-IR spectrum was observed in both model compounds and erythrocyte membranes undergoing peroxidative damage. The feature corresponds to a C-O stretching and/or O-H in-plane bending vibration and is suggestive of the formation of these functional groups during peroxidation. FT-IR provides an accurate means for monitoring the effects of peroxidative damage in human erythrocytes.

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Homozygous protein C (PC) deficiency is a rare genetic defect that usually results in fatal thrombotic complications (purpura fulminans and DIC), but it can be successfully managed with oral anticoagulants or PC replacement. The successful use of PC replacement for two individuals is described. The activity and antigen levels of PC in fresh frozen plasma (FFP) and prothrombin complex concentrate (PCC) are also reported. The concentration of PC in FFP is 87 +/- 15 units/dl. PC is present in all PCC analyzed; however, a ten-fold difference between the various brands and/or lots is noted. The PC activity and antigen correlates well with no significant levels of APC. Upon infusion of FFP into two homozygous PC-deficient children, the PC levels obtained were less than or equal to 30 units/dl post-infusion and undetectable after 12-18 hr. With infusions of PCC, plasma levels of PC obtained were 100-145 units/dl and less than 10 units/dl after 48 hr. The percent recovery and half-lives of PC from FFP and PCC were 49.8% and 7.8 hr, and 84% and 7.4 hr, respectively. One infant was treated every 48 hr for 2 years without significant purpura fulminans or DIC complications. The levels of the other PC system components did not change during the infusion of the PC-rich material. Based on this information, a specific replacement protocol has been developed using a PC-rich concentrate. However, several problems may arise with the "less pure" PC-rich concentrates: catheter-tip thrombosis, related large vessel thrombosis and blood-transmitted diseases. With a specific PC concentrate, replacement therapy is a viable alternative for the long-term management/treatment of homozygous PC deficiency.

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Intraerythrocytic vesicles accumulate in the peripheral blood as a result of impaired clearance of these intracellular inclusions by the spleen. The observation that neonates demonstrate an increased percentage of erythrocytes containing these vesicles constitutes the primary evidence supporting the concept that the newborn is functionally hyposplenic. Neonatal erythrocytes also demonstrate an increased propensity to undergo a variety of endocytic processes. We therefore questioned whether the increase in red cell vesicles in the neonate might be the result of increased vesicle formation as opposed to impaired splenic clearance. Newborn and adult erythrocytes were incubated in vitro in synthetic medium at 37 degrees C. Several parameters confirmed the maintenance of physiologic conditions, including levels of erythrocyte phosphate metabolites monitored by nuclear magnetic resonance. The acquisition of intraerythrocytic vesicles during the course of these incubations was compared. Over a period of 144 h, 19.2% of neonatal erythrocytes acquired vesicles compared to 3.7% of the adult cells (p less than 0.001). The increase in vesicles was greater in younger density-separated erythrocytes in both the neonate (37.6%, p less than 0.0005) and the adult (10.3%, p less than 0.002), but persisted even in the oldest erythrocytes (12.2% and 2.4%, respectively). We conclude that the increase in erythrocytic vesicles in the neonate may not simply be an indication of hyposplenism, but a reflection of increased vesicle formation which overwhelms the clearance capability of the spleen.

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The ability of mature erythrocytes to spontaneously form intracellular vesicles has been implied from clinical studies but has not been examined experimentally. An in vitro model was developed to demonstrate whether mature erythrocytes are capable of spontaneously forming intracellular vesicles. Normal human erythrocytes were incubated in vitro at 37 degrees C for 144 hr in a synthetic medium. During the course of these incubations, approximately 4% of erythrocytes developed intracellular vesicles which were quantitated by using interference contrast microscopy. Electron microscopic studies confirmed the intracellular nature of these vesicles. Incubation of erythrocytes in autologous plasma produced similar results. The rate of vesicle acquisition in vivo was measured by quantitating erythrocyte vesicles immediately prior to and following splenectomy. The rates of vesicle acquisition in vivo and in vitro were comparable. This in vitro model confirms the ability of mature erythrocytes to spontaneously form intracellular vesicles and strongly supports the concept that this is a physiologic process.

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Pentoxifylline was shown to prevent sickling induced by deoxygenation of SS-genotype blood from sickle cell disease patients. It also prevents development of cell stiffness, based on decreased membrane fluidity. This effect is related to increased red cell ATP content, decreased potassium loss during sickling and decreased attachment of hemoglobin to the red cell membrane during sickling. There was no effect on osmotic fragility or mean corpuscular volume. There was some increase in pH of blood from patients in vasoocclusive crisis of sickle cell disease under the effect of pentoxifylline. The consideration is discussed that pentoxifylline may prevent vasoocclusive crisis, but may not abort an existing process.

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Red cell deformability was found to be impaired in SS- and SC-genotype and to a lesser extent SA-genotype red blood cells as compared with those of AA-genotype. RA-233 in vitro improved deformability according to a bell-shaped dose-response curve. RA-233 also prevented experimental vasoocclusive crisis in Macaca arctoides. Deoxygenation of SS-genotype red cells resulted in sickle shape transformation, ATP depletion, potassium efflux, and attachment of hemoglobin molecules to the membrane. These changes were prevented by RA-233. Suspending SS-genotype red blood cells in potassium rich tris-buffer also prevented potassium efflux during deoxygenation and also decreased cellular deformability. RA-233 had no effect on osmotic fragility of SS-genotype red blood cells.

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A wide variety of disorders can result in diminished splenic function. The pathophysiology appears to be clearly defined in some instances, such as congenital asplenia and disorders of splenic vascular obstruction or congestion. In others, such as the autoimmune and GI disorders, the mechanism remains poorly defined. Further research is needed. The hyposplenia which occurs in many of these disorders has been associated with an increased risk of life-threatening, overwhelming bacterial sepsis. In other instances, this complication has not been reported. This certainly should not be interpreted to mean that it cannot occur. The risk of septicemia in hyposplenic disorders is rarely above 10 to 15%. In disorders with minimal inhibition of splenic function, the incidence of sepsis would presumably be less than the 1.5% incidence following surgical splenectomy for trauma. Considering these data, a very large number of patients would have to become asplenic before it would be likely that one would develop sepsis. Furthermore, the lack of awareness of the possibility of hyposplenia-related sepsis in many of these disorders may cause such occurrences to go unrecognized. Finally, since the risk of sepsis is probably less in hyposplenic adults as compared to children, studies on adults may underestimate the incidence of this complication in children. Many of the disorders reported to cause hyposplenia in adults have not been noted to do so in children. In instances such as celiac disease, it may take many years for the complication to manifest so that it would be unlikely for a child to manifest hyposplenia during childhood. However, in other instances, not enough children have been studied to be confident that the hyposplenia and its associated risk of sepsis are not complications that occur in children. Hyposplenia-related bacterial septicemia is a catastrophic complication. If a patient develops a disorder that is potentially associated with hyposplenia, the patient should be observed for signs of asplenia in the peripheral blood. If the technique is available, quantitation of red cell pits should be performed. If not, other studies of splenic function such as radionuclide scans should be considered, depending on the incidence of hyposplenia in that particular disorder. If evidence of asplenia develops, pneumococcal vaccine should be administered, penicillin prophylaxis should be considered, significant febrile episodes should be managed aggressively, and probably most importantly, the patient and family should be carefully educated about this complication. Most deaths from hyposplenia-related septicemia are preventable.

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An infant with recurrent purpura fulminans in the first year of life was found to have severe homozygous deficiency of protein C (less than 1% of normal levels). The episodes of purpura fulminans were controlled by infusions of fresh frozen plasma containing protein C. The requirement of frequent plasma infusions, however, eventually resulted in several complications secondary to hyperproteinemia. Factor IX concentrates rich in protein C were then given to maintain adequate levels of the factor while minimizing the amount of extraneous proteins. The patient has remained asymptomatic and free of complications for greater than 10 months while receiving these concentrates every 48 hours.

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Thrombotic thrombocytopenic purpura is a life-threatening disorder which requires immediate therapy. Unfortunately, there are no widely accepted therapeutic recommendations for this rare disorder. The literature contains large numbers of uncontrolled and often uncomparable studies of a variety of therapies used in differing combinations. This article attempts to rationalize the therapy of thrombotic thrombocytopenic purpura based on our current understanding of its pathophysiology. The rationale of each therapeutic modality, including plasma therapy, corticosteroids, vincristine, antiplatelet agents, and splenectomy, is discussed. This is followed by an overview of the clinical results reported in the literature for these individual treatments. Finally, overall therapeutic guidelines for the therapy of thrombotic thrombocytopenic purpura are presented. It is hoped that these guidelines will clarify the treatment of this disorder until more definitive therapeutic studies are available.

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Thrombotic thrombocytopenic purpura is an uncommon, life-threatening disorder that affects older children and adolescents as well as adults. A variety of theories have been proposed to explain its clinical and pathologic manifestations, but the pathophysiology remains poorly understood. It is not even clear whether this disease primarily affects the endothelial cell, the platelet, or both. Most patients have no discernable predisposition to this disease. Our failure to define the pathophysiology of thrombotic thrombocytopenic purpura adequately has hampered our ability to design rational and consistently successful therapy. The present knowledge of this pathophysiology is discussed in detail. The high mortality of this disease necessitates rapid diagnosis so that therapy can be instituted as quickly as possible. The clinical manifestations and diagnostic criteria of thrombotic thrombocytopenic purpura are therefore reviewed.

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In vitro deformability of red blood cells from SS-genotype patients in vasoocclusive crisis of sickle cell disease was significantly improved by the in vitro addition of the xanthine derivative pentoxifylline (Trental). Monkeys transfused with discarded sickle cell crisis blood showed aggregation of red cells and platelets on metal screens inserted into arterio-venous anastomoses. This phenomenon could be prevented by pretreatment of the animals with 24 mg/kg of pentoxifylline. The possible mechanisms of action are discussed.

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Splenic function in children with hemoglobin SC disease and sickle ß-thalassemia was evaluated using direct interference contrast microscopy. Children with both disorders demonstrated significant degrees of hyposplenism as evidenced by increased numbers of erythrocytes containing surface "pits." Patients with these variants of sickle cell disease frequently exhibit splenic dysfunction and may be at increased risk to develop the life-threatening septicemias associated with hyposplenism.

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We evaluated 100 children with normoblastemia and 400 control children in order to determine the significance of this finding in a pediatric population. None of the control children demonstrated nucleated erythrocytes (NRBC) in their peripheral blood. Among the 100 children with normoblastemia, 49% had underlying disorders associated with hypoxia. Only 8% of the children had malignancies. Eighty-eight percent of the patients in this study had obvious underlying disorders which accounted for their normoblastemia. However, those patients without an obvious etiology for their NRBC were at considerable risk for having bone marrow replacement.

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