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 100-150mg/dL, and 2 points for a level below 100mg/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 150mg/dL and scoring up to 2 points for a level below 100 mg/dL.

Evaluation of the Abbott Cell-Dyn Sapphire Hematology Analyzer / 대한진단검사의학회지

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 withinrun 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 / 대한진단검사의학회지

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.

A Patient with IgA Monoclonal Gammopathy Presenting as Myelomatous Pleural Effusion with Axillary Node Involvement

Multiple myeloma is a cancer of plasma cells that produce monoclonal immunoglobulin, and the neoplastic plasma cells typically accumulate in the bone marrow with occasional involvement of other organs. Pleural effusion that is associated with multiple myeloma has been infrequently reported (<6%) and myelomatous pleural effusion is extremely rare (<1%). A 73-year-old woman was admitted to the department of dermatology for skin lesions on both arms and both ankles. A chest radiograph taken on admission showed a nodular lesion in the right upper lung and pleural effusion. Analysis of the pleural fluid revealed many atypical plasma cells, elevated levels of IgA (27.95g/L) and lambda light chain (9.16g/L), and monoclonal IgA-lambda paraprotein on immunofixation. The serum concentrations of IgA were elevated (33.08g/L) while the concentrations of IgG and IgM were decreased. Bone marrow aspirate smears contained increased levels of immature-appearing atypical plasma cells. This is only the third case of myelomatous pleural effusion that has been reported in Korea.

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.

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-1beta, 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.

Annual Report on External Quality Assesment in Hematology in Korea (2004) / 임상검사와정도관리

Four trials of external quality assessment in diagnostic hematology were performed in 2004 with about 440 participating laboratories in Korea. We performed quality assessment for white blood cell count, hemoglobin, red blood cell count, platelet count, white cell differential count, red blood cell morphology and coagulation test. The response rate was more than 96%. The coefficients of variation in hemoglobin and RBC number was stable but variable in platelet number and WBC number according to measuring cell counts. Blood coagulation study was performed twice. Test results show wide variation according to measuring machine and reagents.

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.

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.

Annual Report on External Quality Assesment in Hematology in Korea (2003) / 임상검사와정도관리

Four trials of external quality assessment in diagnostic hematology were performed in 2003 with about 430 participating laboratories in Korea. We performed quality assessment for white blood cell count, hemoglobin, red blood cell count, platelet count, white cell differential count, red blood cell morphology and coagulation test. The response rate was more than 95%. The performance of quality assessment appeared to be gradually improved year by year.

Annual Report on External Quality Assessment in Diagnostic Genetics in Korea (2003) / 임상검사와정도관리

The importance of quality control for dramatically growing genetic tests continues to be emphasized with increasing clinical demands. Diagnostic genetics subcommitee of KSQACP performed two trials for cytogenetic study in 2003. Cytogenetic surveys were performed by 33 laboratories and answered correctly in most laboratories except some problems in nomenclature and analysis for FISH and complex cytogenetic abnormalities in neoplasia. The molecular genetic test surveys include M. tuberculosis, HBV, HPV, leukemia/lymphoma, ApoE genotyping, Duchenne muscular dystrophy, myoclonic epilepsy and ragged red muscle fibers, and spinal and bulbar muscular atrophy. HPV, myoclonic epilepsy and ragged red muscle fibers, and spinal and bulbar muscular atrophy were the first challenge of the genetic survey. Molecular genetic survey showed excellent results in most participants, however, HPV tests should be improved by quality control in a few laboratories. External quality assessment program for cytogenetic analysis could be helpful to give participants many chances of continuous education and of interesting case materials.

Annual Report on External Quality Assesment in Hematology in Korea (2002) / 임상검사와정도관리

Four trials of external quality assessment in diagnostic hematology were performed in 2002 with about 400 participating laboratories in Korea. We performed quality assessment for white blood cell count, hemoglobin, red blood cell count, platelet count, reticulocyte count, white cell differential count, and red blood cell morphology test. The response rate was more than 90%. The performance of quality assessment appeared to be gradually improved year by year.

Annual Report on External Quality Assessment in Diagnostic Genetics in Korea (2002) / 임상검사와정도관리

The importance of quality control for dramatically growing genetic tests continues to be emphasized with increasing clinical demands. Diagnostic genetics subcommitee of KSQACP performed two trials for cytogenetic study in 2002. Cytogenetic surveys were performed by 33 laboratories and answered correctly in most laboratories except some problems in nomenclature and analysis for mosaicism and cytogenetics of neoplasia. The molecular genetic test surveys include M. tuberculosis, HCV, HBV, leukemia/lymphoma, ABO genotyping, ApoE genotyping, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), and mitochondrial encephalomyopathy, lactic acidosis, and stroke like episodes (MELAS). HBV, SCA, SMA, MELAS tests were the first challenge of the genetic survey. Molecular genetic survey showed excellent results in most participants, however, ABO genotyping tests should be improved by new methods in a few laboratories. External quality assessment program for diagnostic genetics could be helpful to give participants many chances of continuous education and of interesting case materials.

A Case of Gray Platelet Syndrome / 대한혈액학회지

The gray platelet syndrome (GPS) is a rare platelet storage pool deficiency with variable degrees of reduction in the numbers and contents of alpha granules. We report a case of GPS in a two month-old male patient who was admitted to the hospital because of congenital biliary atresia and hip dislocation under the impression of ARC (arthroglyposis, renal tubular dysfunction, and cholestasis) syndrome. Bleeding time was not prolonged and platelet count was normal. The peripheral smear demonstrated abnormal platelet morphology with many agranular and large forms. A platelet aggregation study was abnormal in response to adenosine diphosphate and collagen. Electron microscopy of platelets revealed marked decrease in alpha granules and increase in vacuoles. The patient underwent Kasai operation with mild complication of postoperative bleeding.

Detection of Novel C4517G (Ser743Trp) Mutation in a Family with Type 2A von Willebrand Disease / 대한혈액학회지

Quantitative von Willebrand disease (VWD) are divided into partial deficiency (type 1) and total deficiency (type 3). Qualitative VWD are devided further into four subcategories (2A, 2B, 2M, 2N) based upon the major mechanism by which von Willebrand factor (VWF) function is impaired. Type 2A is characterized by the absence of large molecular weight VWF multimers and a number of mutations have been identified in the region encoding the A2 domain of VWF where a normal cleavage site is situated. Here, we report a case of type 2A VWD in a 5 year-old girl with a novel C4517G (Ser743Trp) mutation, which was also detected in her mother.

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.

Clinical Usefulness of Circulating Hepatocyte Growth Factor (HGF) in Breast Cancer / 대한진단검사의학회지

BACKGROUND: The hepatocyte growth factor (HGF) is a cytokine modulating epithelial cell proliferation and motility. A circulating HGF level is frequently increased in a variety of tumors including advanced breast cancer. The clinical usefulness of measuring circulating HGF in breast cancerwas evaluated in this study. MATERIALS AND METHODS: The plasma HGF levels in patients with primary (n=58) and recurrent breast cancer (n=13) were measured by the ELISA method using Quantikine (R&D, Minneapolis, MN, USA) kit. The results were compared with those of age-matched healthy controls (n=53). The mean (+/-SD) levels of HGF were also compared between primary and recurrent breast cancerpatients. The correlation of the circulating HGF level and the conventional prognostic factors such as tumor size, lymph node involvement, Her-2/neu over-expression, DNA aneuploidy was studied to evaluate the clinical usefulness of HGF as a new prognostic indicator in breast cancer. RESULTS: Plasma HGF levels (mean+/-SD) increased in breast cancer patients (788+/-853 pg/mL), compared with those of age-matched healthy control women (426+/-120 pg/mL) (P<0.05). Patients with recurrent breast cancer (1, 945+/-1, 544 pg/mL) showed increased HGF levels compared with primary breast cancer (592+/-132 pg/mL) (P<0.001). No significant correlations between plasmaHGF levels and conventional prognostic indicators of breast cancer including tumor size, lymphnode involvement, Her-2/neu over-expression, DNA aneuploidy, and histologic grade were found. CONCLUSIONS: The above findings suggest that the measurement of plasma HGF levels in breast cancer patients may be useful for early detection of recurrence.

Altered Expression of Lewis Antigen on Tissue and Erythrocytes in Gastric Cancer Patients

To elucidate the clinical significance of phenotypic alterations of Lewis antigen in gastric cancer patients, we investigated Lewis antigens by analyzing the genotypes of the Le and Se genes and by comparing the results obtained with the phenotypic expression of Lewis antigen in gastric cancer tissue and blood cells. One hundred and twenty gastric cancer patients were examined and compared with respect to Lewis blood phenotype and genotype. The expression of Lea, Leb, sialylated Lea, and sialylated Lex antigens was immunohistochemically examined in uninvolved gastric mucosa, intestinal metaplasia, and cancerous tissue. We also analyzed the significance of Lewis antigen expression by analyzing patient survival. The frequencies of the Lewis phenotypes of RBCs corresponding to Le(a+b-), Le(a-b+), and Le(a-b-) were 16%, 58%, and 26%, respectively. The Le and le allele gene frequencies calculated from genotyping in gastric cancer patients were 0.623 and 0.377, respectively. The frequency for Le(a-b-) of the RBC phenotype had a tendency to be higher in cancer patients than in normal healthy Koreans. However, no difference in the Lewis gene frequency was found between these gastric cancer patients and healthy persons. The phenotype of Le(a-b+) was most prevalent in uninvolved gastric mucosal tissue, whereas the most prevalent form in tumor tissue was Le(a-b-). Sialyl-Lea and sialyl-Lex antigens were hardly detectable in uninvolved gastric mucosa, whereas the two antigens were expressed highly in intestinal metaplastic mucosa and tumor cells. In conclusion, the loss of Lewis antigen expression in tissue and on RBCs in gastric cancer patients is not a result of genetic influences, but rather a result of sialylation in tissue. We also confirm that poor prognosis is associated with dimeric sialyl-Lex and vascular spread.

Evaluation of HER2/neu Status by Real-Time Quantitative PCR in Breast Cancer

Over-expression of the human epidermal growth factor receptor-2 (HER2/neu) has been observed in many cancers, and is associated with a poor prognosis. Recent adjuvant treatment with anti-HER2 monoclonal antibodies in breast cancer has increased the demand for an evaluation of the HER2/neu status in breast cancer. The aim of this study was to investigate the HER2/neu status in breast cancer by a real-time quantitative polymerase chain reaction (PCR) method using LightCycler (Roche Diagnostics, Mannheim, Germany). DNA samples from the fresh tumor tissues of 27 patients with breast cancer were analyzed in parallel using immunohistochemistry (IHC) and the other prognostic parameters including estrogen receptor, progesterone receptor, cytokeratin, and DNA ploidy. Ten (37%) out of 27 cases tested were positive for HER2/neu, while 16 (73%) out of 22 tested positive through an IHC study. The correlation between the DNA aneuploidy and the positive results for HER2/neu were only observed using the real-time PCR method (p < 0.05). There was no significant correlation between the HER2/neu status and the S-phase fractions of the DNA ploidy or other parameters. This study demonstrated that there is marked discordance in the results for the HER2/neu status according to the various methods used. Real-time quantitative PCR for HER2/neu appears to be clinically useful due to its simplicity and ability to produce rapid results.