Kidney-limited thrombotic microangiopathy in patients with SLE treated with romiplostim.

We present the case of a 19 year-old Caucasian female with history of systemic lupus erythematosus (SLE) and normal baseline kidney function who developed severe acute renal failure following treatment of thrombocytopenia with the thrombopoietic agent romiplostim. Percutaneous kidney biopsy revealed thrombotic microangiopathy (TMA) without immune complex lupus glomerulonephritis. We discuss pathogenesis and differential diagnosis of TMA in patients with SLE and raise concerns regarding the use of thrombopoietic agents in such patients. Based on favorable long-term outcome in our case aggressive treatment and in particular prolonged use of plasma exchange in these patients are advocated.

Iodinated contrast media: effect of osmolarity and injection temperature on erythrocyte morphology in vitro.

BACKGROUND: Some side effects of intravenously injected iodinated contrast media are thought to be linked to the biological properties of the various agents and their effect on blood components. PURPOSE: To assess the effect of osmolarity and injection temperature of iodinated contrast media on erythrocyte (RBC) morphology in vitro. MATERIAL AND METHODS: Blood from 20 volunteers was incubated with three different contrast media (320 mg I/ml iso-osmolar iodixanol, 300 mg I/ml low-osmolar iopromide, 300 mg I/ml low-osmolar iopamidol) injected at 37 degrees C, 43 degrees C, and 48 degrees C, and in two different volumes corresponding to the estimated concentration at the site of venous injection and after systemic distribution. After 10 min incubation, aliquots were removed for complete blood count analysis and blood smears. Two hematologists blindedly and independently reviewed all smears, and determined the grade of morphological RBC changes compared to a blank sample. RESULTS: There was excellent (kappa = 0.98) inter-reader correlation for grading RBC changes. At systemic concentration at 37 degrees C, the grade of RBC changes was significantly (P<0.05) less in blood samples exposed to iso-osmolar iodixanol (mean 0.21) as compared to low-osmolar iopromide (mean 0.26) and low-osmolar iopamidol (mean 0.58). These differences became more significant at higher volumes, corresponding to concentrations at the site of injection and higher injection temperatures. CONCLUSION: In vitro, RBC morphology is less affected by iso-osmolar as compared to low-osmolar contrast media. These differences become more significant at higher injection temperatures that are proposed to improve flow dynamics for high-speed injection.

Immunoradiometric measurement of the factor VIII procoagulant antigen.

A fluid-phase immunoradiometric assay has been developed which identifies an antigen on the Factor VIII (antihemophilic factor) procoagulant protein. This sensitive and quantitative assay is not influenced by levels of Favor VIII-related antigen (von Willebrand factor) or other plasma proteins. There is a close correlation of procoagulant activity and immunologically detectable protein in normal and von Willebrand's disease plasmas. In contrast, several different patterns have been identified in hemophilic plasmas. Neither procoagulant activity nor procoagulant antigen is detectable in plasmas from patients with severe classic hemophilia. Patients with mild and moderate hemophilia have either comparable plasma concentrations of procoagulant activity and procoagulant antigen or relatively greater levels of immunologically detectable protein.

The properties of immune complexes formed by human antibodies to factor VIII.

Although human antibodies to Factor VIII inactivate its procoagulant activity, they do not form immunoprecipitates when tested with this antigen. To understand this observation, we have examined the interaction of normal human Factor VIII with four high-titer human anti-Factor VIII, two from transfused hemophiliacs and two "spontaneous" antibodies from nonhemophilic individuals. An estimate of the size of complexes formed by these antibodies has been obtained by agarose gel filtration of mixtures of anti-Factor VIII with cryoprecipitate. Complexed anti-Factor VIII was detected by the method of Allain and Frommel: acid dissociation of complexes at pH 3.5. Complexed anti-Factor VIII was detected in column fractions eluting between the void volume and those which correspond to the elution volume of human IgG. In contrast, Factor VIII procoagulant activity was restricted to void volume fractions when separations were carried out in antigen excess, and free anti-Factor VIII was limited to late-eluting fractions when separations were carried out in antibody excess. A small proportion of the complexed anti-Factor VIII was present in void volume fractions; the quantity was directly related to the ratio of antibody to antigen.Thus, although some complexed anti-Factor VIII is detected in void volume fractions, as would be expected for complexes formed with a very large plasma protein, most immune complexes elute in fractions that indicate interaction with a smaller antigen. These findings suggest that human anti-Factor VIII inactivates procoagulant activity by forming a complex with a small, apparently univalent, component of Factor VIII. This property may prevent immunoprecipitate formation.

Elevated glucose concentrations increase factor VIIIR:Ag levels in human umbilical vein endothelial cells.

To determine if endothelial cell metabolism is affected by the elevated glucose concentrations known to occur in diabetes mellitus, we measured cellular Factor VIIIR:Ag in endothelial cells grown under conditions of increased glucose concentration. The results of this study illustrate that, under cell culture conditions, an increase in glucose concentration results in increased cellular levels of Factor VIIIR:Ag. Specifically, a glucose concentration of 300 mg/dl resulted in a 23% increase in Factor VIIIR:Ag levels while a glucose concentration of 600 mg/dl resulted in a 54% increase in Factor VIIIR:Ag levels. These in vitro findings may relate to the increase in plasma Factor VIIIR:Ag levels known to occur in diabetic patients.

Hemostatic alterations with exercise conditioning in NIDDM.

Various parameters of coagulation and fibrinolysis were measured in 13 men (aged 54 +/- 3 yr) with non-insulin-dependent diabetes mellitus (NIDDM) before and after 12-14 wk of exercise training. Subjects exercised for 30 min 3 times/wk at 70% of maximum O2 consumption (VO2max). Training increased VO2max by 12.5% but did not alter body weight, relative body fat, blood pressure, cholesterol, triglycerides, or high-density lipoprotein cholesterol. Slight downward trends were apparent for fasting glucose and insulin, but glycosylated hemoglobin was unchanged. There were no changes in coagulation parameters of plasminogen, hematocrit, or alpha 2-antiplasmin. Plasma fibrinogen (303 +/- 24.2 vs. 256 +/- 12.3 mg/dl) and fibronectin (380 +/- 41.9 vs. 301 +/- 22.2 micrograms/ml) were significantly reduced (P less than 0.02) by exercise conditioning. Three assays of fibrinolytic activity (tissue plasminogen activator, euglobulin lysis time, and an isotopic measure of fibrinolysis) confirmed that neither basal fibrinolysis nor the fibrinolytic responses to venous occlusion and maximal exercise were significantly altered. Exercise conditioning may have antithrombotic effects in NIDDM by reducing plasma fibrinogen and fibronectin. Although the significance of the fall in fibronectin awaits further studies, the reduction in plasma fibrinogen gives a rationale for the use of exercise training in men with NIDDM.

Platelet-associated IgG assay using flow cytometric analysis.

In this study we describe an immunofluorescent assay system to measure platelet-associated immunoglobulin G levels using flow cytometric analysis. This semi-quantitative system allows ready distinction of immune from non-immune related thrombocytopenias. It is simple to perform, highly reproducible and has the advantage of requiring a minimum concentration of 5000 platelets/microliter per assay sample.

The effect of bacteremia on automated platelet measurements in neonates.

Platelet volume measurements have been used to differentiate consumptive and hypoplastic thrombocytopenia. Since thrombocytopenia is a frequent complication of neonatal sepsis, the authors explored the utility of correlating mean platelet volume (MVP) and platelet distribution width (PDW) with bacteremia. In a sample of 156 infants, there was a significantly increased presence of bacteremia in those infants with MPV greater than 10.8 fL and/or PDW greater than 19.1%. High MPV and PDW showed high specificity for detecting bacteremia (95% and 79%, respectively), and had good negative predictive value. Neonates with blood cultures positive at birth (early infection) tended to have normal platelet volumes, while those infected after three days of age (late infection) had dramatic increases in MPV and PDW. Changes in MPV and PDW should be noted when the diagnosis of late sepsis is considered.

Platelet dysfunction in Noonan's syndrome. A case with a platelet cyclooxygenase-like deficiency and chronic idiopathic thrombocytopenic purpura.

Individuals with Noonan's syndrome are likely to have one or more coagulation abnormalities: complex platelet function defects, partial Factor XI deficiency, or von Willebrand's disease. A distinctive platelet function defect has not been identified. The authors describe a 24-year-old women with Noonan's syndrome, chronic idiopathic thrombocytopenic purpura (ITP), and a platelet function defect characterized by a greater than 15-minute bleeding time, failure of aggregation and release with 10 microM ADP, 10 microM epinephrine, 750 microM arachidonic acid or 0.019 g/L collagen. A mixture of aspirin-treated platelets with the patient's platelets failed to correct the defect. Addition of 2.5 microM U46619 (a PGG2 analogue) corrected the aggregation and release defect. An electron microscopic analysis failed to reveal structural abnormalities. Thus, the platelet function defect in this patient appears to be a functional deficiency of cyclooxygenase. The presence of autoantiplatelet antibodies in a clinical setting consistent with chronic ITP raises the possibility that the defect may be acquired.

Systemic mast cell disease with marrow and splenic involvement associated with chronic myelomonocytic leukemia.

Systemic mast cell disease (SMCD) has a highly variable clinical expression and course. That SMCD is associated with hematologic disorders has been widely described. We report an unusual case of systemic mast cell disease and concurrent chronic myelomonocytic leukemia in a 60 year old male.

Common variable immune deficiency syndrome associated with lupus anticoagulant.

A patient with an acquired immune deficiency syndrome developed a lupus anticoagulant in which the inhibitory activity was manifested only as a prolongation of the partial thromboplastin time. Contrary to previous reports, the substitution of platelets or platelet sonicates for phospholipid in this coagulant assay failed to correct the abnormality and the inhibitor did not exhibit immunoreactivity against phospholipids. The possible mechanisms of action of these inhibitors are discussed.

Acquired dysfibrinogenemia secondary to mithramycin toxicity.

A 58-year-old black woman with IgD multiple myeloma developed a hemorrhagic diathesis within 48 hours after receiving mithramycin (20 micrograms/kg/day) for therapy of hypercalcemia. Her coagulation studies were characterized by prolonged prothrombin, partial thromboplastin, thrombin, and reptilase clotting times. Her plasma and partially purified fibrinogen were inhibitory to the clotting of normal plasma and fibrinogen. The patient's isolated fibrinogen showed a normal rate of fibrinopeptide release, but her fibrin monomer aggregation was markedly abnormal. These studies document the development of a dysfibrinogenemia secondary to mithramycin toxicity.

The role of apheresis in the support of life-threatening ITP relapse.

A 14-year-old girl with chronic idiopathic thrombocytopenic purpura (ITP) presented in relapse with a platelet count of 1,000/microL and a high-level serum antiplatelet IgG antibody. She previously had been unresponsive to courses of therapy with steroids, vincristine, and splenectomy. When treatment with danazol and purified immunoglobulins was unsuccessful in controlling her rapidly progressive course, an 8-day plasma exchange procedure was initiated in combination with platelet transfusion therapy and immunosuppression with cyclophosphamide and vincristine. Within 2 days, her clinical state improved markedly, correlating with a drop in her serum antiplatelet antibody level. She continued to improve and was discharged on a regimen of cyclophosphamide and danazol. Her antiplatelet antibody level had fallen to within the normal range, despite a typical platelet count of 5,000/microL during the 8-day period. Two weeks later her platelet count rose to 65,000/microL. This case suggests that a course of therapeutic plasma exchange may have a temporizing role in the acute management of life-threatening chronic ITP relapse, generating time for the more definitive therapy of immunosuppression to take effect.

The effect of the physicochemical state of factor VIII on its interaction with human antibodies.

To assess the effect of the physicochemical state of factor VIII on its interaction with human antibodies, this interaction was examined using buffer conditions which would insure dissociation of the factor VIII complex. Anti-factor VIII titers were increased 2 to 2.5 fold under such conditions. When isolated factor VIII procoagulant material was used as the antigen source, the antibody titers were increased but no difference was noted in the presence or absence of the high ionic strength buffers. These studies indicate that factor VIII-related antigen in some manner retards the interaction of the procoagulant portion of factor VIII with human antibodies and may explain the peculiar time-dependence of factor VIII inactivation by these antibodies.

The mechanism of factor VIII inactivation by human antibodies. II. The effect of factor VIII R: antigen on the rate of interaction.

Factor VIII R:Ag by binding to the procoagulant component (VIII:C) inhibits the extent and rate of interaction of human antifactor VIII antibodies with VIII:C. When this reaction is examined under ionic strength conditions (0.24M CaCl) which dissociate the two factor VIII components, the extent of the reaction is increased approximately two fold and the initial rate of interaction is increased three to four fold for both intact IgG antibody and its Fab' derivative. With isolated procoagulant component, increased ionic strength conditions only influence the rate of interaction. These studies further explain the peculiar time-dependence of this interaction.

The laboratory diagnosis of lupus anticoagulants.

With the well-documented association of lupus anticoagulants with thrombotic disease and recurrent spontaneous abortion, the laboratory approach to diagnosing these inhibitors is more critical now. To this end, we examined plasma samples from 21 patients who initially presented with a prolonged prothrombin time or activated partial thromboplastin time or both for the presence of lupus anticoagulants. We used a battery of coagulation tests, including both immediate and two-hour mixing studies, a platelet neutralization procedure, a tissue thromboplastin inhibition test, and dilute Russell viper venom times. Two patients (10%) had only a prolonged prothrombin time, seven (33%) had only a prolonged activated partial thromboplastin time, and in 12 (57%) both were abnormal. In 15 patients, inhibition was evident on immediate assay of equal-volume mixture studies of patient plasma and normal pooled plasma, but in three additional patients it was evident only after a two-hour incubation. Fifteen of 18 samples showed correction of the abnormal screening study when platelets were used as a source of phospholipid. Both the tissue thromboplastin inhibition test and dilute Russell viper venom times were sensitive assays, being abnormal in 20 of 21 and 13 of 14 samples, respectively. In four patients, discordance of studies necessitated specific coagulation factor levels being measured to confirm the presence of the inhibitor. Because of the variable effect of the inhibitors on all currently available assay procedures, we would suggest that any evaluation will require a laboratory to have a battery of tests available before such an inhibitor can be excluded.

HLA-Dr negative acute non-lymphocytic leukemia.

Absent or diminished HLA-Dr antigen representation on the cell surface of both normal and leukemic promyelocytes is a hallmark of this stage of myeloid maturation. In order to document the specificity of this finding for acute promyelocytic leukemia, flow cytometric analysis of leukemic blasts was utilized on 36 cases of acute non-lymphocytic leukemia. All 15 of the promyelocytic leukemias (FAB-M3) studied showed absent or markedly decreased HLA-Dr antigen on their cell surface. However, the majority of cases (21) in which this finding was noted were other than promyelocytic leukemias and included all FAB subtypes, most particularly FAB-M2, i.e., myeloblastic leukemia with maturation. It is concluded that absent to decreased HLA-Dr antigen representation on leukemic blasts lacks specificity and can be seen in all acute myeloid/monocytic leukemic subtypes.

Acquired von Willebrand's disease following bone marrow transplantation.

A 41-year-old male underwent allogeneic bone marrow transplantation for the treatment of acute myelogenous leukemia. Six months later, he was admitted to a hospital with signs and symptoms consistent with worsening chronic graft-vs-host disease. Despite a negative past history for a bleeding diathesis, the patient was found to have absent factor VIII procoagulant and ristocetin cofactor activities with markedly reduced von Willebrand factor antigen, all consistent with a diagnosis of acquired von Willebrand's disease. Successful treatment of this disorder with aggressive apheresis and von Willebrand factor replacement therapy is noted.

Mechanism of factor VIII inactivation by human antibodies. IV. Antibody binding prevents factor VIII proteolysis by thrombin.

Factor VIII activation by thrombin is the result of a proteolytic cleavage of the procoagulant component. These studies examine the effect of human antibody on this activation step in a solid phase immunoadsorbent assay system. Radiolabeled factor VIII antibody: factor VIII protein immune complexes were bound to agarose beads by mouse monoclonal antifactor VIII R:Ag antibody. The incubation of these bound labeled immune complexes with high ionic strength buffers (1 M NaCl, 0.24 M CaCl2), or with acidic buffers (0.01 M glycine-0.1 M NaCl, pH 3.0 or 3.5), or with trypsin (1, 5, and 20 mg per ml) dissociated 14 to 62 percent of the bound radiolabel. Thrombin at a concentration of 0.05 U per ml, however, only dissociated 2.9 percent of the label, an amount not significantly different than borate buffered saline control. It is concluded that inactivation of factor VIII is the result of human antibody inhibition of thrombin-induced proteolysis of factor VIII procoagulant protein.

Mechanism of factor VIII inactivation by human antibodies. III. Proteolytic cleavage of factor VIII:C and C antigen by thrombin.

Thrombin activation of factor VIII results in a marked, but transient, increase in factor VIII procoagulant activity. This proteolytic process has been examined by immobilizing factor VIII in a solid phase system using mouse monoclonal antibody specific for the factor VIII related antigen. These studies demonstrate that thrombin activation is the result of proteolytic cleavage from the factor VIII:C protein of a peptide fragment which contains both the factor VIII:C procoagulant and VIII:C antigen sites recognized by human antibodies.