[Evaluation of the Abbott Cell-Dyn Sapphire hematology analyzer].

BACKGROUND: The performance of Cell-Dyn Sapphire (Abbott Diagnostic, USA) was compared to the Bayer Advia 2120 (Bayer Diagnostics, USA), Sysmex XE-2100 (Sysmex Corporation, Japan), and reference microscopy. METHODS: Three hundred samples for routine CBC and WBC differentials were randomly chosen for a comparison analysis. The Cell-Dyn Sapphire system was evaluated according to the linearity, imprecision, inter-instrument correlations, and white blood cell differential. RESULTS: The CBC parameters (WBC, RBC, hemoglobin and platelet) showed a significant linearity with correlation coefficients greater than 0.99 (P<0.0001). Coefficients of variation (CV) for within-run and differential count of WBC were less than 5% except for Total CV for monocytes, eosinophils, and basophils and within-run CV for low valued eosinophils. The correlation coefficients with manual count were lower in monocytes, eosinophils, and basophils than in neutrophils and lymphocytes. The correlation with other hematology anlayzers was significant exclusive of basophils. CONCLUSIONS: These results demonstrate that the Cell-Dyn Sapphire has a good linearity, an acceptable reproducibility, a minimal carryover, and a comparable performance with the sysmex XE-2100 and Advia 2120.

Investigation of von Willebrand factor gene mutations in Korean von Willebrand disease patients.

BACKGROUND: We intended to find the mutations of von Willebrand factor (VWF) gene as the most important contributing factor of von Willebrand disease (VWD) in Korean patients. METHODS: In 40 known vWD patients mutations of vWF gene were sought by direct sequencing of PCR products targeting exons 18, 19, 20, 26, 28 and 52 frequently implicated as the locations of mutation. For factors other than VWF gene contributing to VWD phenotype, we tested ABO blood group and measured ADAMTS13 activity in VWD patients. RESULTS: Twenty-seven cases (67.5%) were type 1 vWD, 3 cases (7.5%) type 3, and 5 cases (12.5%) type 2A. Three cases were type 2A or 2B (7.5%) and 2 cases were suspected to be type 2N (5.0%). Among them six candidate missense mutations were found: V1279I, R1306W, R1308C, and V1316M were previously reported in type 2B and type 1 vWD, and C858W and T1477I were novel findings. All patients were heterozygotes. Blood group O was overly represented in VWD patients, while ADAMTS13 activity of the patients was not significantly different from that of normal control. CONCLUSIONS: Mutation of VWF gene detected by genetic studies can significantly improve the diagnostic accuracy, especially in subtype assignment of VWD. Two novel mutations, C858W and T1477I associated with VWD were found and expected to contribute to the elucidation of its pathophysiology.

Diagnosis of overt disseminated intravascular coagulation: a comparative study using criteria from the International Society versus the Korean Society on Thrombosis and Hemostasis.

PURPOSE: Since 1993, Koreans have used diagnostic criteria set by the Korean Society on Thrombosis and Hemostasis (KSTH) in the diagnosis of overt disseminated intravascular coagulation (DIC). In 2001, the Scientific and Standardization Committee (SCC) of the International Society on Thrombosis and Hemostasis (ISTH) proposed new diagnostic criteria for the diagnosis of overt DIC. We wanted to compare the use of the ISTH versus KSTH criteria in the diagnosis of overt DIC. MATERIALS AND METHODS: We enrolled 131 patients over the age of 15 years, who had been admitted and diagnosed as having DIC from May 2000 to April 2005 at the Youngdong Severance Hospital, Seoul, Korea. Of the 131 patients, there were 71 males and 60 females, with a median age of 61 years. Hemostatic tests, including platelet counts, PT, aPTT, fibrinogen level and D-dimer, were evaluated based on the respective scoring systems. To assess the concordance between the two diagnostic systems, we used the Student's t-test and the K-coefficient. RESULTS: There were 79 patients compatible with the ISTH criteria and 63 patients with the KSTH criteria. Sixty-one patients were compatible with both diagnostic systems. The grade of agreement, or concordance rate, was 84.7% and the K-coefficient, or interrater reliability, was as low as 0.6 without significance. However, if we scored 1 point for a fibrinogen level of 100-150 mg/dL, and 2 points for a level below 100 mg/dL, for the ISTH criteria, then 63 patients were compatible with both diagnostic systems, and the concordance rate increased to 85.5% and the K-coefficient to 0.71 with significance. CONCLUSION: To achieve good agreement between the ISTH and KSTH diagnostic systems for overt DIC, we highly recommend changing the plasma fibrinogen cut-off value in the ISTH criteria from 100mg/dL to 150 mg/dL and scoring up to 2 points for a level below 100 mg/dL.

Fibrinogen Seoul (FGG Ala341Asp): a novel mutation associated with hypodysfibrinogenemia.

Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in fibrinogen function. The molecular basis of hypodysfibrinogenemia was investigated in a 66-year-old woman with peripheral artery obstructive disease and in her family members. Plasma level of functional fibrinogen determined using the Clauss method was lower (75 mg/dL; normal, 140-460 mg/dL) than that measured with immunologic nephelometric assay (137 mg/dL; normal, 180-400 mg/dL). Similar results were also observed in two family members through two generations. DNA was extracted from whole blood, and the coding regions and intron/exon boundaries of gamma chain gene (FGG) were amplified. A novel (Fibrinogen Seoul) heterozygous FGG mutation (GCT->GAT, Ala341Asp) was identified in all three affected family members. Thrombin-catalyzed polymerization was found to be defective on the analysis of purified fibinogen from the propositus. Molecular modeling also showed a conformational change of fibrinogen structure.

A novel fibrinogen variant (fibrinogen Seoul II; AalphaGln328Pro) characterized by impaired fibrin alpha-chain cross-linking.

We report a novel fibrinogen variant (fibrinogen Seoul II), which has a heterozygous point mutation from CAA to CCA leading to AalphaGln328Pro. The mutation site is among several glutamine residues that serve as alpha-chain cross-linking acceptor sites. Fibrinogen Seoul II was found in a 51-year-old male patient and his family in Seoul, Korea. The patient was diagnosed with myocardial infarction at age 43. Eight years later he was admitted to the emergency room due to recurrence of the disease, where he expired under treatment with tissue plasminogen activator (t-PA). Fibrin polymerization curves, made using purified fibrinogen from the patient's relatives, showed a decreased final turbidity, suggesting Seoul II fibrin clots are composed of thinner fibers. This supposition was verified using scanning electron microscopy. Alpha-polymer formation by the mutant fibrinogen upon thrombin treatment in the presence of factor XIII and calcium was distinctly impaired. This result confirms that the residue Aalpha328 plays a pivotal role in alpha-chain cross-linking.

Plasma factor XIII activity in patients with disseminated intravascular coagulation.

The objective of this study was to investigate the correlation between factor XIII (FXIII) activity and disseminated intravascular coagulation (DIC) parameters and also to evaluate the clinical usefulness of DIC diagnosis. Citrated plasma from eighty patients with potential DIC was analyzed for FXIII activity. The primary patient conditions (48 male and 32 female, mean age, 51 years) were malignancy (n = 29), infection (n = 25), inflammation (n = 6), heart disease (n= 3), thrombosis (n = 2), injury (n = 2), and other miscellaneous conditions (n = 13). FXIII testing was performed using the CoaLinkTM FXIII Incorporation Assay Kit (PeopleBio Inc.). Among 80 patients who were suspected to have DIC based on clinical analysis, 46 (57.5%) fulfilled the overt DIC criteria (DIC score > = 5) according to the International Society of Thrombosis and Haemostasis. FXIII levels in the plasma were significantly decreased in overt DIC compared to non-overt DIC patients (mean 75.1% and 199.7% respectively, p < 0.0001). Interestingly, we found a significant inverse correlation between DIC scores and FXIII activity. In addition, FXIII activity significantly correlated with other hemostatic markers that included platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen, and D-dimer. FXIII levels were significantly lower in patients with liver or renal dysfunction. In conclusion, FXIII cross-linking activity measurements may have differential diagnostic value as well as predictive value in patients who are suspected to have DIC.

Plasma level of IL-6 and its relationship to procoagulant and fibrinolytic markers in acute ischemic stroke.

Procoagulant or impaired fibrinolytic states as well as inflammatory reactions mediated by cytokines are likely involved in the pathogenesis of acute ischemic stroke. We examined the potential relationship between interleukin 6 (IL-6) and hemostatic markers. The procoagulant and fibrinolytic states were assessed in 46 patients with acute stroke by measuring plasma levels of plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and plasminogen-antiplasmin complex (PAP). Circulating IL-6 levels were measured using ELISA (Quantikine, R and D systems, MN, USA). Circulating IL-6 (mean, 26.5 pg/mL) and PAI-1 (mean, 19.9 ng/mL) levels were higher in patients with acute stroke than in healthy subjects (mean, 3.0 pg/mL, 10.4 ng/mL, respectively). TAT levels were statistically different according to the etiologic subtypes of stroke (atherogenic, 2.5 ng/mL; lacunar 3.2 ng/mL; cardiogenic 9.9 ng/mL, p = 0.021). Neither procoagulant levels nor fibrinolytic markers significantly correlated with circulating IL-6 levels. Our findings suggest that elevated proinflammatory cytokines during the initial hours of ischemic stroke may be an independent pathogenic factor or a consequence of the thrombotic event with no relationship to the procoagulant or fibrinolytic states.

Effect of plasma exchange on the circulating IL-6 levels in a patient with fatal hemophagocytic syndrome associated with bile ductopenia.

We report here the case of a patient suffering from hemophagocytic syndrome (HPS) associated with bile ductopenia. A 24-year old man was admitted after suffering fever, sore throat and general malaise for 7 days and jaundice for 2 days. Clinical studies showed hepatic dysfunction with hyperbilirubinemia. Epstein-Barr viral DNA from two bone marrow samples was detected. Bone marrow aspiration disclosed findings of HPS. Liver biopsy showed centrilobular cholestasis with lack of interlobular bile duct. Repeated therapeutic plasma exchange was effective for decreasing serum bilirubin and interleukin-6 levels. The patient received liver transplantation, however, he finally died of alveolar hemorrhage resulting from disseminated intravascular coagulation and acute rejection.

Novel monoclonal antibody that recognizes new neoantigenic determinant of D-dimer.

Our novel monoclonal antibody (mAb) B4 reacted with only D-dimer but not intact fibrinogen, or fibrinogen degradation products (FgDP) such as D-monomer, E fragment on ELISA. B4 didn't react with denatured D-dimer, while it reacted well with denatured D-monomer rather than the native form, indicating that B4 recognizes some neoconformational epitope in D-dimer. In our epitope study, B4 recognized the N-terminal (Bbeta134-142) of D-dimer, which corresponds to the most flexible segment of coiled coil backbone. It was confirmed by inhibition assay of B4 binding to D-dimer using the synthesized peptides with this sequence. As the other evidence, B4 didn't bind to some D-dimer species produced from a particular fibrinogen variant. This fibrinogen variant is mutated BbetaLys133 residue to Gln133 thus it doesn't produce the particular N-terminal epitope of D134 approximately by plasmin. Finally, our mAb was useful for clinical application. ELISA using our mAbs was well correlated with other commercial D-dimer ELISAs and in some clinical samples it was preferable to them. These results suggest that the epitope for B4 is another neoantigenic determinant in native D-dimer as distinct from native D-monomer.

Upstream regulation of matrix metalloproteinase by EMMPRIN; extracellular matrix metalloproteinase inducer in advanced atherosclerotic plaque.

From experimental and clinical studies it is known that matrix conservation and degradation by matrix metalloproteinases (MMPs) plays a major role in plaque progression and destabilization with related onset of acute vascular events such as acute coronary syndromes or cerebrovascular accidents. Recently, extracellular MMPs inducer (EMMPRIN) has been reported to induce and activate the expression of MMPs in myocardium and plays an important role in the ventricular remodeling in human heart failure. Similarly to heart failure myocardium, EMMPRIN may be expressed in human atheroma and play a role in the extracellular matrix (ECM) remodeling and atherogenic cell differentiation. This study was designed to investigate the possible biological role of EMMPRIN in human atheroma. Immunohistochemical analysis for MMPs and EMMPRIN was performed on human carotid endarterectomy specimens and control aortas. EMMPRIN showed significant immunoreactivity in human atherosclerotic carotid lesions, and was colocalized with macrophage/monocyte infiltrates in atherosclerotic intima, plaque itself and vascular smooth muscle cells (VSMCs). Zymography and Western blot analysis revealed EMMPRIN expression in the carotid atheromas, but not in the control aortas. Human bone marrow monocytes, which were cultured with atherogenic proinflammatory cytokine stimulation revealed increased EMMPRIN and MMPs expressions. ECM remodeling is under the control of induction and inhibition of matrix degrading protease and the novel MMP inducer, EMMPRIN may play a role in influx and differentiation of monocytes and destabilizing atheroma.

Isolation of endothelial progenitor cells from cord blood and induction of differentiation by ex vivo expansion.

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1 beta , fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.

Tissue transglutaminase autoantibodies in patients with IgM rheumatoid factors.

The recent identification of tissue transglutaminase (tTG) as the autoantigen for celiac disease-associated anti-endomysial antibodies (EMA) has allowed the use of rapid immunoassay to detect the presence of autoantibodies, anti-tTG, in the serum of patients. In this study, we examined the prevalence of IgG or IgA anti-tTG in sera from patients with elevated levels of IgM rheumatoid factors, which are autoantibodies reactive with the Fc portion of IgG. We report here on four cases of anti-tTG positivity for patients with elevated IgM rheumatoid factor (RF) without evidence of celiac sprue. The study population consisted of 65 patients (26 men, 39 women; mean age, 49 years; range 4 - 92 years) with elevated RF (>20 U/ml ), and 23 healthy subjects (12 men, 11 women; mean age, 46 years; range, 21 - 54 years). IgG and IgA anti- tTG levels were detected using a commercially available ELISA kit (Immuno-Biological Laboratories, Germany). Out of 65 patients, one (1.5%) and three (4.6%) patients were positive for IgG and IgA anti-tTG antibodies, respectively, and this was a higher frequency than occurred in healthy subjects (0/23). The clinical features of the four cases positive for IgG or IgA anti-tTG were as follows: The first case (female, 63 yrs) positive for IgA anti-tTG antibody suffered from rheumatoid arthritis, type II diabetes mellitus, iron deficiency anemia and gastric indigestion without symptoms of malabsorption. She denied any gluten sensitivity on her diet. Her esophagogastroduodenoscopic biopsy showed mucosal atrophy with no elongated crypts or infiltration of inflammatory cells in the lamina propria. The remaining three cases positive for anti-tTG antibodies had interstitial pneumonia, a herniated lumbar disc, and mild scoliosis, respectively. They all denied any malabsorption symptoms or gluten sensitivity. Jejunal biopsy could not be performed in all four cases.

Comparison of plasma D-dimer and thrombus precursor protein in patients with operable breast cancer as a potential predictor of lymph node metastasis.

Fibrin formation and removal is continuous during the development of malignancy. Plasma D-dimer is indicative of ongoing fibrinolysis, and circulating soluble fibrin polymer [thrombus precursor protein (TpP)] represents thrombogenic activity. We evaluated the relationship between plasma D-dimer and TpP levels with tumor extent and examined the use of these markers as possible predictors of lymph node metastasis in breast cancer. Preoperative plasma levels of D-dimer and TpP were measured in these 120 patients (93 breast cancer, 27 benign breast disease) and 29 healthy controls. Plasma levels of D-dimer in patients with breast cancer were significantly higher than in healthy controls and in those with benign breast disease. Plasma D-dimer levels in patients with breast cancer were found to be significantly increased according to tumor stage. We also observed that plasma levels of D-dimer were higher in patients with lymph node metastasis than in patients without metastasis. In contrast, TpP levels were not significantly different by the tumor stages and lymph node metastasis. In conclusion, increased D-dimer levels in breast cancer may suggest that an ongoing fibrinolysis within breast cancer tissue occurs during tumor progression. Positive D-dimer levels might be useful for identifying metastatic lymph node in patients with operable breast cancer. However, plasma TpP was not found to be a sensitive marker for detecting tumor-associated subclinical coagulopathy.

Prevalence of beta2-glycoprotein I antibody in patients with liver cirrhosis: relationship with beta2-glycoprotein I plasma levels and thrombosis.

Recently, a semiquantitative enzyme-linked immunoassay for detecting IgG or IgM class autoantibody to beta2-GPI for use as an aid in the diagnosis of certain autoimmune or thrombotic disorders. We evaluated the prevalence of anti-beta2-GPI antibody and its relation to plasma beta2-GPI levels and occurrence of thrombosis in 69 patients with liver cirrhosis. As expected, plasma beta2-GPI levels were lower in the patients with liver cirrhosis and correlated with Child classification. Anti-beta2-GPI antibodies were positive in 11 (15.9%) patients. However, there were no differences in beta2-GPI levels, platelet counts, and thrombosis between positive and negative groups for anti-beta2-GPI antibodies.

Plasma levels of tissue factor antigen in patients with non-insulin-dependent diabetes mellitus.

Patients with diabetes mellitus (DM) are associated with an increased risk of thrombosis, and are susceptible to a series of complications including nephropathy. It has also been known that plasma tissue factor (TF) antigen levels increase significantly in certain disease states. To investigate the clinical significance of an association with the various complications in patients with type 2 non-insulin-dependent DM (NIDDM), we measured the plasma levels of TF antigen in 63 patients (35 males and 28 females, mean age 60.8 yrs) with NIDDM and in 22 normal subjects (14 males and 8 females, mean age 56.0 yrs). The mean concentrations of TF were higher for patients with NIDDM (253.7 +/- 144.9 pg/ml) than in normal subjects (187.3 +/- 108.7 pg/ml with marginal statistical significance (p= 0.0530). The TF levels were higher for patients with a nephropathy than for patients without a nephropathy (p=0.0402). There was a significant positive correlation between levels of TF and BUN (r=0.84, p < 0.0001) or creatinine (r=0.93, p < 0.0001). However, TF levels were found to be similar for both groups with and without thrombosis, neuropathy, retinopathy, or infection. These results suggest that plasma TF antigen levels may be associated with nephropathy and they may reflect a renal dysfunction in NIDDM.

Platelet microparticles induce angiogenesis in vitro.

Platelet microparticles (PMP) are endogenous substances generated during the coagulation process in a hypercoagulable state. This study demonstrated that PMP promote the proliferation and survival, migration, and tube formation in human umbilical vein endothelial cells (HUVEC). Heat-treated PMP did not significantly decrease the angiogenic activity in HUVEC compared with that of the untreated PMP. Meanwhile when PMP were treated with activated charcoal, a procedure known to remove the lipid growth factors, the angiogenic activity was significantly reduced. These results suggest that the lipid component(s) of the PMP may be major active factor(s) and that protein component(s) may be minor contributor(s). PMP were also shown to augment endothelial progenitor cell differentiation in peripheral blood mononuclear cells. In addition, PMP-stimulated proliferation, chemotaxis and tube formation of the HUVEC was mediated via the Pertussis toxin-sensitive G protein, extracellular signal-regulated kinase and the phosphoinositide 3-kinase pathway. Herein, a new action of PMP was demonstrated to be a potent angiogenic stimulator. It is expected that in pathological states such as a growing tumour, PMP shed from the circulating platelets may reach adequate concentrations and that the elevated levels of PMP could contribute to florid formation of new blood vessels.

Circulating numbers of endothelial progenitor cells in patients with gastric and breast cancer.

Angiogenic factors like VEGF or G-CSF were reported to mobilize endothelial progenitor cells (EPCs) from the bone marrow. These EPCs were shown to be incorporated in the neovessels of developing tumors. Although the concentrations of angiogenic factors in the peripheral blood were reported to be elevated in cancer patients, the number of circulating EPCs has not been previously investigated. In this study, the number of EPCs circulating in the blood in 16 healthy controls and 71 newly diagnosed cancer patients was examined by a culture assay of peripheral blood mononuclear cells. The number of circulating EPCs was not found to be increased in cancer patients, although the plasma levels of VEGF were elevated. It is suggested that VEGF, at concentrations typical of those observed in the blood of cancer patients, does not mobilize EPCs into the peripheral blood.

Detection of an Ala601Thr mutation of plasminogen gene in 3 out of 36 Korean patients with deep vein thrombosis.

Plasminogen is a key proenzyme in the fibrinolytic and thrombolytic systems. Congenital deficiency of plasminogen and molecular abnormality of plasminogen (dysplasminogenemia) have been reported in association with the thrombotic tendency in human. In dysplasminogenemia, the level of immunoreactive plasminogen is normal, although the functional activity is reduced. Human plasminogen gene spans about 52.5 kb of DNA and consists of 19 exons. Three types of mutations (Ala601Thr, Val355Phe, and Asp676Asn) have been described in dysplasminogenemia. In this study, we measured the plasminogen activity in patients with deep vein thrombosis and analyzed the DNA sequence to detect three point mutations (Ala601Thr, Val355Phe and Asp676Asn) in patients with hypo/dysplasminogenemia. Dysplasminogenemia was identified in 3 (8.3%) of unrelated 36 patients with deep vein thrombosis and the Ala601Thr mutation was detected in all three patients with dysplasminogenemia. In conclusion, dysplasminogenemia is not rare in deep vein thrombosis, which suggests a risk factor for the thrombosis in Korean population.