Lawrence S. Lessin

Plasma factor VII and thrombin–antithrombin III levels indicate increased tissue factor activity in sickle cell patients

Although the mechanisms involved in the persistent clinical complications of sickle cell disease have not yet been fully delineated, previous studies suggest that sickle cell (HbSS) patients have a disposition to generate more thrombin and plasmin in vivo than normal subjects. The reasons for the impaired regulation of haemostasis in HbSS patients is poorly understood. We report studies evaluating the extent to which in vivo coagulation and fibrinolysis are altered in HbSS patients in steady state. The concentrations of total factor VII (F(VII)t), factor VII zymogen (F(VII)z), thrombin–antithrombin III (TAT), fibrinopeptide A(FPA), and fibrin D‐dimer in plasmas of 50 normal controls (HbAA) and 45 HbSS steady state patients, were measured using sensitive and specific enzyme‐linked immunoassays. The average plasma concentration of F(VII)t, in sickle cell plasma was significantly lower than that of the control subjects (0·70 ± 0·19 U/ml versus 1·16 ± 0·41 U/ml), whereas F(VII)z in the patients and controls were 0·47 ± 0·15 U/ml and 1·15 ± 0·33 U/ml respectively, P<0·001. Both measures of factor VII suggest a higher factor VII turnover in sickle cell disease. The mean concentration of TAT in the plasma of HbSS patients were significantly higher than those of HbAA controls (371 ± 44 pM versus 42 ± 2 pM) (P<0·001), a difference that is strongly indicative of higher rates of in vivo thrombin generation by HbSS patients. Plasmas of HbSS patients had significantly higher concentrations of FPA compared to those of the control subjects (12·85 ± 1·96 ng/ml versus 4·22 ± 0·37 ng/ml) (P<0·001). The D‐dimer levels were also higher in the HbSS than control plasmas (1029·6 ± 58·6 ng/ml versus 224·3 ± 27·6 g/ml) (P<0·001), with the patients’values being indicative of enhanced fibrinolysis. These results strongly suggest accelerated in vivo coagulation and fibrinolysis in HbSS patients even during steady state. They are consistent with the hypothesis that haemostasis is less tightly regulated in the HbSS patients than in HbAA controls. The altered regulation of haemostasis may contribute to the initiation of vaso‐occlusive processes associated with sickle cell painful episodes.

Bone-Marrow Mast Cells in Lymphoproliferative Disorders

Increased bone-marrow mast-cell content and lymphoproliferative disorders have been previously linked. Using a semiquantitative method we examined bone-marrow mast-cell content in 120 marrow specimens from patients with multiple myeloma, chronic lymphocytic leukemia, non-Hodgkins lymphoma, and reactive lymphocytosis. Results indicated a statistically significant increase of marrow mast-cell content in patients with chronic lymphocytic leukemia, non-Hodgkins lymphoma, and reactive lymphocytosis when compared with iron-deficient control subjects (p less than or equal to 0.0005). Patients with multiple myeloma had decreased marrow mast-cell content, clearly separating them from patients with lymphoproliferative disorders and reactive lymphocytosis. Linear regression plot of marrow mast-cell content against percentage of marrow lymphocytes showed a direct relation, indicating that marrow mast-cell density may be related more to the degree of lymphoid proliferation than to the specific lymphoproliferative process. Marrow mast-cell content may therefore be reproducibly determined and used to support the morphologic diagnosis of lymphoproliferative disorders and differentiate them from atypical myelomas.

Bone marrow mast cell content in preleukemic syndrome.

Bone marrow mast cell content was evaluated by a semiquantitative method in 22 marrow specimens from 20 patients with preleukemic syndrome and was compared with 21 marrow specimens from iron-deficient control subjects. Results indicate a statistically significant increase of bone marrow mast cell content in patients with preleukemic syndrome in comparison to control subjects (p less than or equal to 0.0005). Two of 20 preleukemic patients converted to acute myeloblastic leukemia and conversion was accompanied by a significant decrease of bone marrow mast cell content. Our findings indicate that bone marrow mast cell content can be reproducibly quantitated and represents an additional morphologic criterion for diagnosis of the preleukemic syndrome.

Flow dynamics of human sickle erythrocytes in the mesenteric microcirculation of the exchange-transfused rat

To analyze the microvascular rheology of sickle cells in an intact animal model, rats were isovolemically exchange transfused with human normal (hemoglobin AA) or sickle (hemoglobin SS) erythrocytes (blood group O) or autologous red cells under ambient conditions, and the effects of the heterologous or autologous cells on (a) hemodynamics and respiration, (b) blood gases, and (c) acid-base status of the recipients were determined. Exchange transfusion of rats with autologous red cells or hemoglobin AA or hemoglobin SS erythrocytes was associated with stable mean arterial blood pressure, central venous pressure, respiration rate, blood pH, pCO2, and pO2 during the experimental period, except for tachycardia among the group of rats that received HbSS cells. Arteriovenous oxygen content varied among the three groups of animals, but, nonetheless, suggested adequate tissue oxygen supply under the conditions of the study. Acid-base status also was similar in the three groups of rats. The exchange-transfused rats were utilized to investigate the flow dynamics of red cells in the mesenteric microcirculation by applying intravital microscopy. Time-averaged velocities of the autologous red cells in 16- to 30-microns (id) vessels ranged from 1.07 to 1.25 mm/sec in single unbranched arterioles with varying flux and wall shear rates. Time-averaged velocities of the HbAA cells in single 15- to 35-microns arterioles ranged from 1.16 to 1.24 mm/sec with wall shear rates similar to the estimates for the autologous cells. For both rat and human HbAA RBCs, the flow dynamics were indicative of normal shear-dependent and deformability characteristics of the cells under the flow conditions. Sickle cells exhibited time-averaged velocities of 0.384 to 0.452 mm/sec, lower wall shear rates in 10- to 35-microns single unbranched arterioles, and three times less volumetric flux. In some arterioles, sickle cells with high axial ratio and low deformability showed definite adhesion to the endothelial surface, residing at such sites for several seconds until dislodged by the force of flow. Within single unbranched vessels or at microvascular bifurcations, sickle elliptocytes and sickle echinocytes with low deformability and high axial ratio obstructed flow and exhibited residence times of 2 to 88 sec, thereby causing stasis. These data illustrate the microvascular flow behavior of sickle cells and demonstrate the rheological disequilibrium state that can result as sickle cells course through successive segments of the microcirculation.

Deformability of Normal and Sickle Erythrocytes in a Pressure-flow Filtration System

In order to measure resistance of erythrocyte suspensions to flow through nuclepore filters, a positive pressure cell filtration system has heen developed and applied to measure the deformability of erythrocytes from normal (AA) individuals and patients with sickle cell trait (AS), sickle cell anemia (SS) and hereditary spherocytosis (HS). The pressure flow filtration system permits manipulation of flow rate (Q), hematocrit, pO2, pH, filter pore size and other metabolic parameters. Erythrocyte deformability is defined in this system as the reciprocal of “relative resistance” (Rr) to flow of a given cell suspension. Resistance is derived as the quotient of the pressure generated in response to a specific flow rate; and Rr is the ratio of actual resistance due to the cell suspension to the resistance of the medium alone. Utilizing this system, the following observations have been made: 1. Increase in hematocrit produces an increase in Rr particularly at low flow rates with a direct relationship between hematocrit and resistance until the cell concentration exceeds the filter pore density, resulting in an exponential rise in the pressure generated by increase in Q 2. With 5 μ filters, at low Q and under ambient pO2, AA and AS cells show slight increase in Rr, whereas, SS and HS cells show almost 10-fold greater Rr; 3. Plots of Rr vs. Q simulate a standard viscometric plot and thus data derived from this positive pressure filtration system is comparable to data obtained from cone-plate viscometry; 4. Reduction of pO2 from 165 to 50 mm Hg produces no effect on Rr vs. Q for normal cells, moderate increase for AS cells and marked increase for SS cells; 5. Scanning electron microscopy of the Nucleopore filters fixed during filtration flow depicts cell deformation and pore occlusion by various cell suspensions providing a morphologic basis for the observed resistance-flow kinetics.

Ultrastructure of the Normal and Hemoglobinopathic Red Blood Cell Membrane: Freeze-Etching and Stereoscan Electron Microscopic Studies

The ultrastructure of the red blood cell membrane and its surfaces is characterized by freeze-etching and stereoscan electron microscopy for the normal red blood cell and cells from patients with sickle cell anemia, hemoglobinopathy CC, and Heinz body hemolytic anemia. The external surface, intramembrane surfaces, and the internal surface of the normal red blood cell are described and the alterations of membranocytoplasmic relationships in the hemoglobinopathies studied are shown.

Plasma and urinary leukotrienes in sickle cell disease: possible role in the inflammatory process

Abstract. Sickle cell (HbSS) disease is associated with rheological and inflammatory stresses within the microcirculation. In order to determine the role of leukotrienes in the inflammatory processes in HbSS patients, we analysed plasma and urine levels of leukotrienes (LT); LTB4, LTC4, LTD4, and LTE4 as indicators of their in vivo metabolism. Plasma and urine level samples of 15 HbSS patients in steady‐state and age‐matched healthy, homozygous (HbAA) controls were extracted for leukotrienes and quantitated by HPLC. Control plasma level of leukotrienes (mean ± SEM, ng ml‐1) were: LTB4, 8·95 ± 0·26; LTC4, 7·24 ± 0·21; LTD4, 11·42 ± 0·40; and LTE4, 14·51 ± 0·50. Corresponding values for HbSS patients were: LTB4, 6·15 ± 0·42; LTC4, 13·61 ± 1·45; LTD4, 6·44 ± 0·51 and LTE4, 4·97 ± 0·37. The differences were significant at P < 0·05. Urine levels (mean ± SEM, ng mmol‐1 creatinine), for controls were: LTB4, 10·60 ± 0·35; LTC4, 360·0 ± 9·82. Values for HbSS urine were: LTB4, 27·50 ± 3·33; LTC4, 356·0 ± 17·87; LTD4, 69·90 ± 14·51. LTD4 was not detected in control urine. These results suggest that sickle cell patients may exhibit impaired ability to catabolize LTC4 in plasma during steady state conditions. This altered metabolism may contribute to the persistent stress of the microcirculation, and is probably related to the abnormal microvascular rheology of sickle blood cells.

Amphotericin-B-induced thrombocytopenia.

Excerpt Despite its toxicity amphotericin B is the drug of choice for treatment of most fungal infections. The known adverse effects, including anemia, hypokalemia, hypomagnesemia, and renal insuff...

Vaso-occlusion in sickle cell disease: pathophysiology of the microvascular circulation.

Microvascular dysfunction accounts for the major morbidity and contributes to the mortality among patients with sickle cell hemoglobinopathies. We summarize the microcirculatory dynamics of red cells in sickle cell disease. An overview of the physiological attributes of the microcirculation is presented. The microcirculatory module is a unique organic entity within the tissue domain, which is concerned with the functional exchange of substances between the blood and the tissue environment. The impairment in deformability of sickle red cells and their heterogeneity cause them to show abnormal microvascular flow dynamics that, in turn, contribute to derangement of the microvascular bed. Studies of experimental models in animals have employed the microcirculation of the mesentery, the cremaster muscle, and the mesoappendix. These studies showed the rheological disequilibrium that results as sickle cells course through successive segments of the arterioles, capillaries, and venules. Direct in vivo microscopic observations in human subjects, with analysis and quantitation of the nailfold and bulbar conjunctival capillaries, have also provided useful information as to the adverse effects of sickling on the microcirculation. Sickle cell vaso-occlusion has three phases--initiation, propagation, and resolution. This framework provides a basis for testable hypotheses for verification in appropriately designed experiments. In this context, the determinants of the microvascular flow of erythrocytes in sickle cell disease are emphasized.

Acquired pure red cell aplasia associated with chronic lymphocytic leukemia.

Pure red cell aplasia (PRCA) represents selective failure of erythropoiesis in the absence of abnormalities in leukopoiesis or thrombocytopoiesis. Acquired PRCA has occurred uncommonly in chronic lymphocytic leukemia (CLL). We report six patients with non‐T‐cell CLL in which nine episodes of PRCA were observed. In six of the nine episodes, the patients had received recent oral alkylating agents; conversely, recovery from aplasia occurred in some while on alternate alkylator therapy and two patients had no pretreatment. Spontaneous occurrence and recovery, therefore, could not be excluded. Onset of PRCA may be suggested by macrocytosis, but discontinuance of therapy at its development may not prevent PRCA if, indeed, therapy is one of the causes. Supportive therapy or alkylators and prednisone permitted recovery without mortality in all six patients. The interval between onset of CLL and the recognition of first PRCA episode varied from one month to nine years (mean, 5.3 years); the duration of PRCA ranged from three to nine months (mean, 4.7 months). In addition to previously better understood causes of anemia in CLL, the uncommon occurrence of PRCA is an additional consideration in the differential diagnosis of sudden anemia in CLL.