Clot waveform analysis using CS-2000i™ distinguishes between very low and absent levels of factor VIII activity in patients with severe haemophilia A.

INTRODUCTION: A recently developed method to assess comprehensive coagulation function, clot waveform analysis (CWA), accurately detect low levels (<1 IU/dL) of factor VIII activity (FVIII:C) in haemophilia A patients (HA-pts). Improvements are needed, however, to differentiate patients with very low from absent levels of FVIII:C. AIM: We attempted to optimize CWA using the coagulation analyser CS-2000i™ to distinguish between very low levels and absent FVIII:C in severe HA-pts. METHODS AND RESULTS: Activated partial thrombin time (aPTT)-based clot waveforms were determined in FVIII-deficient plasmas mixed with various amounts of recombinant FVIII. Clot times (CT) were shortened, and maximum coagulation velocity (|min1|) and acceleration (|min2|) were increased in FVIII dose-dependently at levels ranging from 0.25 to 100 IU/dL. The lowest level of FVIII:C detected was 0.25 IU/dL. Plasma samples from modestly severe (MS-HA; 0.5-<1.0 IU/dL), very severe (VS-HA; 0.25-<0.5 IU/dL), extremely severe (ES-HA; <0.25 IU/dL) and inhibitor-positive HA-pts (HA-inh) were examined. The CT was markedly prolonged in all instances but showed significant differences between the different groups insufficiently. The |min1| and |min2| in HA-inh were lower compared to the other groups (P<.05). A new parameter (slope-|min1|) reflecting average coagulation acceleration was derived. This index (median) was lower in HA-inh (0.0042) compared to ES-HA (0.0068) and VS-HA (0.011) with greater significant differences (P<.01), and an index of <.005 reflected the total absence of FVIII in the presence of inhibitor. CONCLUSION: The slope-|min1| parameter could provide a useful index for evaluating very low and absent levels of FVIII and/or the development of FVIII inhibitor in HA-pts.

Emicizumab, the bispecific antibody to factors IX/IXa and X/Xa, potentiates coagulation function in factor XI-deficient plasma in vitro.

Essentials Emicizumab mimics factor (F)VIIIa cofactor function, augments the intrinsic tenase activity. We assessed the emicizumab-driven hemostatic function in FXI-deficient plasmas. Emicizumab improved the coagulation potentials in severe FXI-deficient plasma. Emicizumab may provide a possibility for clinical application in patients with FXI deficiency. SUMMARY: Background Patients with factor (F)XI deficiency commonly present with markedly prolonged activated partial thromboplastin times (APTT), although bleeding phenotypes are heterogeneous. Emicizumab, a bispecific monoclonal antibody to FIX/FIXa and FX/FXa, mimics FVIIIa cofactor function on phospholipid (PL) surfaces. Antibody reactions were designed, therefore, to augment mechanisms during the propagation phase of blood coagulation. Aim To assess emicizumab-driven hemostatic function in FXI-deficient plasmas. Methods and Results Standard ellagic acid (Elg)/PL-based APTTs of different FXI-deficient plasmas (n = 13; FXI activity, < 1 IU dl-1 ) were markedly shortened dose dependently by the presence of emicizumab. To further analyze the effects of emicizumab, clot waveform analysis (CWA) in FXI-deficient plasmas with emicizumab, triggered by tissue factor (TF)/Elg demonstrated improvements in both clot times, reflecting the initiation phase, and coagulation velocity, which represents the propagation phase. Emicizumab also enhanced the TF/Elg-triggered thrombin generation in FXI-deficient plasmas dose-dependently although the degree of enhancement varied in individual cases. Thrombin generation with either FVII-deficient plasma or FIX-deficient plasma treated with anti-FXI antibody showed little or no increase by the co-presence of emicizumab, suggesting that the accelerated thrombin generation in FXI-deficient plasmas by emicizumab should depend on the FIXa-involved coagulation propagation initially triggered by FVIIa/TF. The ex vivo addition of emicizumab to whole blood from three patients with severe FXI deficiency demonstrated modest, dose-dependent improvements in Ca2+ -triggered thromboelastograms (NATEM mode). Conclusion Emicizumab appeared to improve coagulation function in severe FXI-deficient plasma, and might provide possibilities for clinical application in patients with FXI deficiency.

Casein hydrolysate formula-induced liver dysfunction in a neonate with non-immunoglobulin E-mediated cow's milk allergy.

A 10-day-old male neonate was admitted with bilious vomiting and gross hematochezia. Peripheral eosinophilia, delayed positive skin prick test to artificial milk, and elevated eosinophil cationic protein levels suggested cow's milk allergy. Fluid infusion with prohibition of oral intake improved the digestive symptoms. Breast-feeding was resumed on hospital day 3 and only casein hydrolysate formula was fed from day 7 onward. Nevertheless, eosinophilia and elevated transaminase levels developed on day 14. Liver dysfunction associated with casein hydrolysate formula was suspected and the infant was transferred to soy formula. Eosinophil counts decreased and transaminase levels were normalized on day 19. A cow's milk protein-specific lymphocyte proliferation test was positive for alpha-casein, beta-lactoglobulin, and bovine serum albumin, indicating sensitization of T cells to cow's milk proteins. These observations suggest that careful attention should be paid to liver dysfunction in non-immunoglobulin E-mediated cow's milk allergy, even when hypoallergenic formula is used.

Assessing the clinical severity of type 1 von Willebrand disease patients with a microchip flow-chamber system.

BACKGROUND: The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. METHODS: Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30 ) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. RESULTS: PL-T10 values correlated with BS (R(2) ~ 0.45) better than VWF:RCo (R(2) ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R(2) ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of < 10 IU dL(-1) than in those with VWF:RCo of 10 IU dL(-1) to < 25 IU dL(-1) and 25-40 IU dL(-1). In patients with VWF:RCo of < 10 IU dL(-1) , BS was significantly higher in those with PL-T10 > 8 min than in those with PL-T10 ≤ 8 min. CONCLUSION: T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.

Effect of recombinant human macrophage-colony stimulating factor on marrow, splenic, and peripheral hematopoietic progenitor cells in mice.

The effects of human macrophage colony-stimulating factor (M-CSF) on marrow, splenic, and peripheral progenitor cells (CFU-M, CFU-GM, and CFU-G) were investigated in mice administered recombinant human M-CSF (8-4,000 micrograms/kg). Single injection of 4,000 micrograms/kg of M-CSF resulted in a decrease in the number of marrow progenitor cells (CFU-M, CFU-GM, and CFU-G) on day 2 followed by a gradual increase, returning to the original level on day 4 or 5. In contrast, each type of splenic progenitors tested for started to increase markedly on day 2, reaching a level 4- to 15-fold higher than that of the basal value on day 3 or 4. Peripheral CFU-M, CFU-GM, and CFU-G also increased on day 2. In addition, administration of 800 micrograms/kg of M-CSF in mice caused a decrease in marrow CFU-G, as well as an increase in splenic CFU-G. The present results indicate that treatments of mice with pharmacological concentrations of human M-CSF affect the number of progenitor cells not only of monocyte/macrophage lineage but also of granulocyte lineage. Also, the coincidence between decrease of marrow progenitor cells and increase of splenic and peripheral progenitor cells suggests that the progenitor cells are released from bone marrow to peripheral blood and reseeded to the spleen by the action of M-CSF.

Mild hemophilia A patient with novel Pro1809Leu mutation develops an anti-C2 antibody inhibiting allogeneic but not autologous factor VIII activity.

BACKGROUND: In mild hemophilia A (MHA) patients, the risk of inhibitor development is generally low, but some factor VIII (FVIII) gene missense mutations are associated with a higher inhibitor incidence. OBJECTIVE: To investigate the mechanism(s) of inhibitor development in MHA. METHODS AND RESULTS: A patient, HA78, with MHA with a novel P1809L missense mutation in the A3 domain, exhibited significant residual FVIII activity ( FVIII: C ~10 IU dL(-1) ), despite the development of an inhibitor (5.6 BU mL(-1) ). Purified HA78-IgG significantly depressed FVIII: C from normal plasma but not from patient's plasma without inhibitor, indicating that this IgG inhibited allogeneic but not autologous FVIII. The HA78-IgG blocked thrombin and FXa-catalyzed FVIII cleavage but had little effect on FVIII binding to von Willebrand factor and phospholipid. The IgG recognized a C2 epitope close or overlapping the previously described anti-C2 ESH8 epitope. Similarly, a recombinant FVIII-P1809L mutant was little inactivated by HA78-IgG. This mutant demonstrated ~3-fold lower binding affinities to von Willebrand factor and phospholipid compared with wild-type, while reactions with thrombin or FXa were not impaired. Reaction of FVIII-P1809L with the alternative anti-C2 ESH4 showed only an ~20% inhibition compared with wild-type FVIII but was similar to wild-type after incubation with ESH8. A surface plasmon resonance-based assay demonstrated that anti-C2 ESH4 bound to FVIII-P1809L with ~10(2) -fold lower affinity compared with ESH8. CONCLUSION: These results indicated that the P1809L mutation in A3 induced the conformational change in the FVIII molecule that hampered antigenic determinant(s) located in the C2 domain and might result in the inhibitor development.

Systematic monitoring of hemostatic management in hemophilia A patients with inhibitor in the perioperative period using rotational thromboelastometry.

BACKGROUND: The management of hemophilia A (HA) patients with inhibitors on bypassing therapy remains challenging. In particular, the monitoring of treatment is restricted by the limited reliability and lack of standardization of currently available methods to evaluate the physiological effects of various hemostatic agents. Accurate monitoring of these patients is particularly important in surgical situations. The recently developed comprehensive coagulation assays, including rotational thromboelastometry (ROTEM), may be useful in these circumstances. OBJECTIVE: We have attempted to establish a systematic monitoring protocol using ROTEM (NATEM triggered by CaCl2 ) to evaluate the choice and effectiveness of different bypassing agents in the perioperative period. METHODS AND RESULTS: The hemostatic effects of recombinant factor VIIa (rFVIIa) and activated prothrombin complex concentrates (aPCC) were determined using a three-step procedure (spike, preoperative and perioperative) in eight patients with HA inhibitor admitted for elective surgery and assessed for individually tailored therapy. The ROTEM parameters demonstrated similar improvement to approximately normal levels at each stage after treatment with rFVIIa. Results in the presence of aPCC showed a marked improvement in the spike data, although this appeared to be different from those in the preoperative and perioperative assessments. The information derived from the spike and preoperative findings provided a useful guide for establishing an effective dose of therapeutic material, and facilitated good hemostatic control during and after surgery in all cases. CONCLUSION: The findings suggest that this systematic analysis using ROTEM could provide a promising strategy for the use of bypassing therapy in HA patients with inhibitor.

Effects of macrophage colony-stimulating factor (M-CSF) on lipopolysaccharide (LPS)-induced mediator production from monocytes in vitro.

M-CSF is a macrophage-lineage-specific growth factor that causes proliferation and differentiation of progenitor cells in the bone marrow. To investigate the effects of M-CSF on more matured cells, human monocytes were cultured in the presence or absence of M-CSF for 6 days. Addition of M-CSF at more than 10(2) U/ml resulted in higher viability and caused morphological differentiation to large macrophage-like cells. LPS-induced mediator production was also compared between M-CSF-treated and control cell. Monocytes were incubated with or without M-CSF for 3 days, and were stimulated with 1 microgram/ml of LPS for 2 days. IL-1 beta was not detected in the both culture supernatants, and PGE2 production was not influenced by M-CSF. However, amounts of G-CSF, GM-CSF, IL-6, and TNF-alpha produced in response to 1 microgram/ml of LPS were 1.5 to 2 times greater from monocytes treated with 10(4) U/ml of M-CSF than from control cells. The priming effect of M-CSF on LPS-induced cytokine production was found to require 3-day preincubation, and reached a maximum at the concentration of 10(4) U/ml. M-CSF-treated cells responded to a 10 times lower concentration of LPS than control cells in terms of cytokine production. M-CSF was also shown by flowcytometric analysis to influence the expression of CD14, a receptor for LPS, which might render monocytes more sensitive to LPS.

Effects of recombinant human macrophage colony-stimulating factor on atherosclerotic lesions established in the aorta of high cholesterol-fed rabbits.

Anti-atherosclerotic effects of human macrophage colony-stimulating factor were investigated using rabbits fed a high cholesterol diet. Rabbits fed a diet containing 2% cholesterol for 59 days developed hyperlipidemia and atheromatous aortic plaques. They were then administered 80 microg/kg/day of either macrophage colony-stimulating factor or human serum albumin, as a control, for the next 12 weeks. Compared with the control group, rabbits treated with macrophage colony-stimulating factor had significantly fewer plaques on the inner surface of the thoracic and abdominal aortae, and half the sectional area of thickened intima in the aortic arch, as well as in the thoracic and abdominal aortae. Macrophage colony-stimulating factor also decreased the cholesterol content of the atherosclerotic lesions. Serobiochemical analyses revealed that macrophage colony-stimulating factor increased the levels of high density lipoprotein-cholesterol significantly, without influencing other lipid parameters such as the level of low density lipoproteins. The effects of macrophage colony-stimulating factor were evident until the fourth week of drug injection, at which time anti-human macrophage colony-stimulating factor antibodies were clearly induced in the serum. These results indicate that exogenously administered macrophage colony-stimulating factor suppresses atherosclerotic lesions induced by a high cholesterol diet by activating lipid metabolism in vivo.

Pituitary choristoma composed of corticotrophs and adrenocortical cells in the sella turcica.

A pituitary tumour composed of well-differentiated corticotrophs and adrenocortical cells is reported. Sections of the tumour revealed a mixture of small round cells with amphophilic or basophilic periodic acid-Schiff (PAS)-positive cytoplasm and large spherical and oval cells with abundant, granular, partly vacuolated PAS-negative cytoplasm. The small cells contained type 1 cytokeratin-positive microfilaments, numerous 250-500 nm endocrine-type secretory granules immunoreactive for adenocorticotropic hormone (ACTH) and beta-lipotropin. The large cells possessed ample cytoplasm filled with abundant vesicular smooth endoplasmic reticulum, numerous mitochondria possessing tubulovesicular cristae and frequent dense bodies. They lacked the features of pituitary endocrine cells or folliculostellate cells and were found to contain a panel of steroidogenic dehydrogenases and hydroxylases. The tumour was classified as a choristoma, in which two distinct cells types, corticotrophs and adrenocortical cells, were mixed. We suggest that, under continued ACTH stimulation, umcommitted stem cells may differentiate into adrenocortical cells. Alternatively, the presence of adrenocortical cells may be the result of heterotopia.

Spontaneous carotid cavernous fistula presenting only with cranial nerve palsies.

PURPOSE: To discuss the differences in angiographic findings between cases of spontaneous carotid cavernous fistula with and without the classical triad of symptoms (pulsating exophthalmos, bruit, and conjunctival chemosis). METHODS: With CT, MR, and angiography, we examined 12 cases of spontaneous carotid cavernous fistula, five of whom presented only with cranial nerve palsies. RESULTS: In the seven cases with the triad, the main venous drainage from the cavernous sinus was the superior ophthalmic vein. Only one or two veins drained the cavernous sinus, and cortical venous drainage was not present in any case. In contrast, all but one case with only cranial nerve palsies had at least three venous drainage routes from the cavernous sinus, including cortical venous drainage. CONCLUSION: For the diagnoses of spontaneous carotid cavernous fistula, it is important to know that some patients do not show the classical triad of symptoms. In such patients, early diagnosis and treatment are particularly important because cortical venous drainage and a consequent risk of hemorrhage are frequently present.

An unruptured arteriovenous malformation with edema.

We report a case of unruptured arteriovenous malformation in which an extensive zone of increased signal intensity in the brain parenchyma adjacent to the nidus is demonstrated on T2-weighted MR. This area of perilesional hyperintense signal exerts a compressive effect, suggesting that it represents perilesional edema.

Pathophysiologic assessment of stagnating arteries after removal of arteriovenous malformations.

PURPOSE: To assess the hemodynamics and pathophysiology of stagnating arteries after removal of arteriovenous malformations (AVMs). SUBJECTS: 50 patients with supratentorial pial AVMs underwent pre- and postoperative angiographic studies. RESULTS: The following characteristics were found to correlate with stagnating arteries: 1) advanced patient age, 2) large AVM size, 3) markedly dilated feeders, 4) early postoperative angiograms, and 5) delayed restoration of feeding artery diameter. CONCLUSIONS: The rate of blood flow in the former feeding arteries, expressed as v x pi x r2 (v = mean velocity, r = vessel radius), suddenly decreases after removal of AVMs. When dilatation persists postoperatively in these arteries the flow velocity decreases and stagnation takes place. Delayed postoperative restoration of feeding artery diameter may be caused by a decrease of elasticity due to long-standing hemodynamic stresses, and by increased postoperative vascular resistance of these arteries.

Superselective embolization for severe traumatic epistaxis caused by fracture of the skull base.

Intractable epistaxis developed in a 13-year-old girl after she fell down a flight of stairs sustaining facial fractures and fracture of the skull base. Epistaxis was found to emanate from a right ascending pharyngeal artery, which the authors promptly and successfully embolized using polyvinyl alcohol particles and microfibrillar collagen.