Establishment and characterization of a novel Hodgkin lymphoma cell line, AM-HLH, carrying the Epstein-Barr virus genome integrated into the host chromosome.

We describe the establishment and characterization of a cell line, AM-HLH, obtained from a patient with Epstein-Barr virus-positive (EBV+ ) nodular sclerosis-type Hodgkin lymphoma (HL). The cells were positive for CD2 and CD30 and negative for CD15. The immunoglobulin heavy- and κ light-chain genes were rearranged. The karyotype was of the triploid range. Southern blotting using the EBV terminal repeat probe detected 3 hybridizing bands that were identical to those of the parental HL material. The cells expressed EBV-encoded RNAs as well as latent genes (EBNA1, EBNA2, LMP1, and LMP2A) and lytic genes (BZLF1 and BALF2). Fluorescence in situ hybridization (FISH) with the cosmid pJB8 clone containing a fragment of EBV DNA as a probe revealed multiple hybridization signals at a marker chromosome. Additional FISH using whole chromosome painting and centromere probes in combination with multicolor FISH determined that multiple EBV copies were clustered within the chromosome 20 materials of the marker chromosome. Culture supernatants of AM-HLH contained IL-10 as measured by the bead-based immunoassay. It is possible that an integrated EBV genome and cellular genes on chromosome 20 were coamplified, leading to the enhanced expression of genes involved in cell growth control. The AM-HLH cell line will be useful to clarify the role of cytokines in the development of EBV+ HL.

Delayed Development of Hemolytic Anemia with Fragmented Red Blood Cells and Cardiac and Renal Impairments after High-Dose Chemotherapy and Autologous Hematopoietic Stem Cell Transplantation for Malignant Lymphoma.

Among 42 consecutive patients with malignant lymphoma who underwent high-dose chemotherapy (HDC) followed by autologous hematopoietic stem cell transplantation (AHSCT), 5 developed hemolytic anemia with fragmented red blood cells (HA-FrRBCs) on days 87-125 (median 107) of AHSCT. Nadir Hb levels ranged between 5.0 and 6.4 g/dL with 2.2-5.6% FrRBCs. All patients developed grade ≥3 hypoxia and heart failure, and 4 developed grade ≥3 hypertension. The ejection fraction of the left ventricle assessed by echocardiography was significantly reduced in 3 patients. Peak creatinine levels were >4 times above the baseline and estimated glomerular filtration rates were reduced to <30 mL/min/1.73 m2. One patient received plasma exchange, while the remaining 4 responded to treatment with diuretics and cardiovascular agents. Hematological parameters normalized within a median duration of 91 days after the development of HA-FrRBCs. Renal and cardiac functions gradually improved, even though renal function did not return to the baseline. HA-FrRBCs associated with cardiac and renal impairments may represent a thrombotic microangiopathy syndrome and are a delayed complication of HDC/AHSCT. The close monitoring of laboratory abnormalities and persistent treatment with cardiovascular agents and diuretics are the mainstay for the management of this condition.

The Influence of Assay Selection on Prothrombin Time Measured in Patients Treated With Rivaroxaban for Nonvalvular Atrial Fibrillation.

BACKGROUND: Prothrombin time (PT) can provide a qualitative assessment of the relative intensity of anticoagulation by rivaroxaban. More than ten types of assay are available for the measurement of PT in clinical settings, but it is not yet fully understood whether their interactions with rivaroxaban are uniform or inconsistent. METHODS: We examined 139 blood samples from patients taking rivaroxaban. We measured PT using five different commercially available assays. We also evaluated the estimated rivaroxaban concentration using a chromogenic anti-factor Xa assay. RESULTS: The median estimated concentration of rivaroxaban was 192 ng/ml (interquartile range 85-284 ng/ml). The correlation coefficient (r) between PT and the estimated concentrations of rivaroxaban was as follows: Thromborel S, r = 0.768; Thrombocheck PT, r = 0.861; Coagpia PT-N, r = 0.909; Neoplastin Plus, r = 0.882; and Triniclot PT Excel S, r = 0.870. The gradients of the regression plots differed more than fourfold, and the standard deviation of the regression line ranged from 1.001 to 2.980, which tended to be higher for the assays with the higher regression slope gradients. CONCLUSION: The estimated concentration of rivaroxaban varied greatly depending on the assay, so the PT measured in patients taking rivaroxaban should be interpreted with caution.

Two patients with α-chain hemoglobin variant Hb Q-Iran detected by measuring hemoglobin A1c using the variant mode of the HA-8180V HPLC analyzer.

Hemoglobin variants are often discovered when hemoglobin A1c (HbA1c) levels measured with a high-performance liquid chromatography (HPLC) system in fast mode are found to be low. The HA-8180V HPLC analyzer by Arkray offers two measurement modes: fast mode (FM) and variant mode (VM). Two Japanese patients with α-chain variant Hb Q-Iran detected incidentally after analyses with the HA-8180V in VM showed an abnormal peak, are presented. The first patient was a man in his 70 s, and the second patient was a man in his 50 s. Both were non-diabetic, but their results from HbA1c measurement in VM showed an abnormal peak. The VM-HbA1c, FM-HbA1c, and HbA1c measured by enzymatic assay and glycated albumin levels of the two patients were all within the reference ranges. They were diagnosed as having Hb Q-Iran (α2-75Asp → His) by globin gene analysis. It is difficult to detect α-chain hemoglobin variants based on abnormal FM-HbA1c levels, but measuring HbA1c in VM is useful for efficiently detecting hemoglobin variants.

New method to differentiate between lupus anticoagulants, progressive coagulation inhibitors and coagulation factor deficiencies in the mixing tests.

INTRODUCTION: Mixing tests in activated partial thromboplastin time (APTT) are used for the differentiation between lupus anticoagulants (LA), coagulation inhibitors, and factor deficient samples with APTT prolongation. However, the indexes for the differentiation have not been established. The present study aimed to develop new mixing test indexes for the differentiation. METHODS: Twenty-six LA-positive, 8 progressive coagulation factor VIII inhibitor, and 35 coagulation deficient samples were employed. APTT were measured for normal plasma, patient plasma, and mixing plasma prepared at a ratio of 1:1 proportion in both without incubation and 2 h-incubation. New two parameters named as ALD50 and mixture plasma-patient plasma after Warming change rate Subtraction (WaS) calculated from the clotting times of normal, 1:1 mixing and patient samples with/without 2 h-incubation were established. In the samples with WaS result of <10.2%, ALD50 of ≥87.8%, and < 87.8% were defined as LA and coagulation factor deficiency, respectively, and WaS of ≥10.2% defined progressive coagulation factor inhibitors. RESULTS: Sensitivity and specificity to LA were 80.8% and 93.0% for ALD50, and sensitivity and specificity to progressive coagulation factor inhibitor were 100.0% and 100.0% for WaS, respectively. The agreement between sample classification and WaS-ALD50 was 88.4% (61/69). CONCLUSIONS: ALD50 and WaS showed acceptable sensitivity and specificity to LA and progressive coagulation factor inhibitor, respectively. These indexes would be useful for the differentiation between LA, factor deficiency, and progressive coagulation factor inhibitor in the mixing tests.

Measuring serum prothrombin in patients treated with warfarin or direct oral anticoagulants.

BACKGROUND: Warfarin therapy influences generation of γ-carboxyglutamyl (Gla) residues in prothrombin, causing reduced coagulation activity. It will leave such inactive prothrombin in serum after clot formation, resulting in serum prothrombin constituting total inactive prothrombin in these patients. METHODS: An ELISA was developed to measure biologically inactive prothrombin in serum, and applied to serum from warfarin therapy causing a decrease in Gla residues or direct oral anticoagulant (DOAC) therapy as its contrast. RESULTS: The concentrations of serum prothrombin in both the warfarin and DOAC groups were higher than those in the healthy group (p < 0.01 and p < 0.001, respectively). When serum in the previous three groups was treated with barium carbonate to exclude prothrombin, which lost several Gla residues, the prothrombin concentration in the DOAC group decreased to the same level as that in the healthy group, indicating that prothrombin was obtained at a high level only in the warfarin group (p < 0.01). CONCLUSIONS: Warfarin and DOAC led to increase in serum prothrombin concentration. The reason is that DOAC decreases prothrombin recruitment during fibrin clot formation, while warfarin leads to the accumulation of inactive prothrombin, which have a decreased number of Gla residues.

The First-Derivative Curve of the Coagulation Waveform Reveals the Cause of aPTT Prolongation.

Clot waveform analysis based on activated partial thromboplastin time (aPTT) is reported to be a useful assay. We attempted to find beneficial parameters with the first-derivative curve. We examined 106 plasma samples with prolonged aPTT and analyzed the first-derivative curve statistically by dividing it into 6 groups (Lupus anticoagulant, Heparin, Direct oral anticoagulants, Factor VIII inhibitor, Hepatic dysfunctions and Factor deficiency). We obtained 7 coordinates for parameter measurement by analyzing the first-derivative curve and set 20 parameters including the velocity axis, the time axis, and area parameters. The distribution was checked by extracting each parameter that showed the most significant difference in the 6 groups. As a result, it was revealed that we could classify aPTT prolongation by using a combination of 3 parameters, the initial-to-peak gradient, the ratio initial-to-intermediate velocity/intermediate-to-peak velocity, and the initial-to-peak area size. We constructed a flowchart combining these 3 parameters and were able to discriminate 75% of the specimens. These parameters derived from the first-derivative curve of clot waveform analysis are useful tools to discriminate aPTT prolongation.

Influence of Warfarin Therapy on Prothrombin Production and Its Posttranslational Modifications.

BACKGROUND: Protein induced by vitamin K absence-II (PIVKA-II) is produced by the liver during hepatoma and upon warfarin administration. Those patients have disturbed protein synthesis and glycosylation in the liver. This decreases the number of γ-carboxyglutamyl (Gla) residues on prothrombin, converting prothrombin into PIVKA-II. The mechanism of this conversion, however, is not clearly understood. METHODS: Prothrombin was isolated from healthy and warfarin-treated individuals whose liver function of protein production was quantitatively normal. Glycan structures in the purified prothrombin containing PIVKA-II were qualitatively analyzed by high performance liquid chromatography after labeling the glycan with fluorophore 2-aminobenzamide. RESULTS: The concentration of PIVKA-II was significantly higher in the warfarin-treated individuals than in the healthy individuals (P< 0.001). Although protein production in the liver was normal in both groups, the concentration of prothrombin was lower in the warfarin-treated individuals than in the healthy individuals (P < 0.001). The main glycan was A2 in the healthy and warfarin-treated individuals (86.6 ± 4.4% and 85.6 ± 3.4%, respectively). Eight types of glycan were characterized in both groups, although generation of PIVKA-II in the warfarin-treated individuals did not lead to variation in glycosylation of prothrombin. CONCLUSIONS: Warfarin therapy leads to lower amounts of prothrombin and Gla residues within prothrombin without exerting qualitative and quantitative change in glycan profile and protein synthetic function in the liver.

Acute non-heparin-induced thrombocytopenia during hemodiafiltration in a patient with multiple myeloma.

This report demonstrates that not only heparin-induced thrombocytopenia, but also hemodialysis conditions (platelet activation due to hemodiafiltration and heparin underdosing) may markedly reduce the platelet count and cause clotting in the hemodialysis circuit in patients in a hypercoagulable state. The clot prevention effects of bortezomib are therefore of great importance.

Acute basophilic leukemia associated with the t(16;21)(p11;q22)/FUS-ERG fusion gene.

We herein report a rare case of acute basophilic leukemia with t(16;21)(p11;q22) generating the FUS-ERG fusion gene. The basophilic nature of leukemia blasts was demonstrated by cytomorphology, toluidine blue metachromasia, mature basophil-associated antigen expression, and characteristic granules under electron microscopy. The molecular link between t(16;21)/FUS-ERG and basophilic differentiation remains unclear.

A novel role for Notch ligand Delta-1 as a regulator of human Langerhans cell development from blood monocytes.

Human Langerhans cells (LCs) are of hematopoietic origin, but cytokine regulation of their development is not fully understood. Notch ligand Delta-1 is expressed in a proportion of the skin. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta1 (TGF-beta1) are also secreted in the skin. We report here that Delta-1, in concert with GM-CSF and TGF-beta1, induces the differentiation of human CD14(+) blood monocytes into cells that express LC markers: CD1a, Langerin, cutaneous lymphocyte-associated antigen, CC chemokine receptor 6, E-cadherin, and Birbeck granules. The resulting cells display phagocytic activity and chemotaxis to macrophage inflammatory protein-1alpha (MIP-1alpha). In response to CD40 ligand and tumor necrosis factor alpha, the cells acquire a mature phenotype of dendritic cells that is characterized by up-regulation of human leukocyte antigen (HLA)-ABC, HLA-DR, CD80, CD86, CD40, and CD54 and appearance of CD83. These cells in turn show chemotaxis toward MIP-1beta and elicit activation of CD8(+) T cells and T helper cell type 1 polarization of CD4(+) T cells. Thus, blood monocytes can give rise to LCs upon exposure to the skin cytokine environment consisting of Delta-1, GM-CSF, and TGF-beta1, which may be, in part, relevant to the development of human epidermal LCs. Our results extend the functional scope of Notch ligand delta-1 in human hematopoiesis.

Relationship between plasma dabigatran concentration and activated partial thromboplastin time in Japanese patients with non-valvular atrial fibrillation.

BACKGROUND: Activated partial thromboplastin time (aPTT) is recommended for monitoring anticoagulant activity in dabigatran-treated patients; however, there are limited data in Japanese patients. To clarify the relationship between plasma dabigatran concentration and aPTT, we analyzed plasma dabigatran concentration and aPTT at various time points following administration of oral dabigatran in a Japanese hospital. METHODS: We enrolled 149 patients (316 blood samples) with non-valvular atrial fibrillation (NVAF) who were taking dabigatran. Patients had a mean age of 66.6±10.0 years (range: 35-84) and 66% were men. Plasma dabigatran concentrations and aPTT were measured using the Hemoclot(®) direct thrombin inhibitor assay and Thrombocheck aPTT-SLA(®), respectively. Samples were classified into eight groups according to elapsed times in hours since oral administration of dabigatran. RESULTS: Significantly higher dabigatran concentrations were observed in samples obtained from patients with low creatinine clearance (CLCr) (CLCr<50 mL/min). Dabigatran concentrations and aPTT were highest in the 4-h post-administration range. Additionally, there was a significant correlation between plasma dabigatran concentrations and aPTT (y=0.063x+32.596, r (2)=0.648, p<0.001). However, when plasma dabigatran concentrations were 200 ng/mL or higher, the correlation was lower (y=0.040x+38.034 and r (2)=0.180); these results were evaluated by a quadratic curve, resulting in an increased correlation (r (2)=0.668). CONCLUSIONS: There was a significant correlation between plasma dabigatran concentrations and aPTT. Additionally, in daily clinical practice in Japan, plasma dabigatran concentrations and aPTT reached a peak in the 4-h post administration range. Considering the pharmacokinetics of dabigatran, aPTT can be used as an index for risk screening for excess dabigatran concentrations in Japanese patients with NVAF.

[Two cases of acute lymphoblastic leukemia with cytoplasmic granules].

The presence of cytoplasmic granules in blastoid cells of patients with acute leukemia is generally accepted as a useful morphological marker for differentiation of myeloid leukemia from lymphoblastic leukemia. We diagnosed two cases of acute lymphoblastic leukemia(ALL) with cytoplasmic granulation. Surface marker analysis of leukemic cells revealed they were positive for CD10, 19, 20, 33, 34 and HLA-DR. Immunoglobulin gene rearrangement was detected by means of Southern hybridization with an Ig-JH probe for both patients. On the basis of these findings, the patients were diagnosed as having B-precursor ALL. Electron microscopic observation showed no myeloperoxidase activity, so that the granules were considered to be related to autophagolysosomes. This experience demonstrates that the recognition of the presence of granular ALL is necessary for making an accurate differential diagnosis of acute leukemias.

Short- and long-term effects of rituximab for the treatment of thrombotic thrombocytopenic purpura: four case reports.

We report four cases of thrombotic thrombocytopenic purpura (TTP) successfully treated with rituximab in combination with plasma exchange and other immunosuppressive agents. All four cases fulfilled the diagnostic criteria of TTP with severe deficiencies in ADAMTS13 activity and a detectable anti-ADAMTS13 inhibitor. Four weekly doses of 375 mg/m(2) rituximab were initiated on day 3-29 of presentation as a salvage treatment for relapsing/refractory disease in three patients and as a first-line treatment in one. Resolution of clinical symptoms and hematological abnormalities occurred as early as the second dose and, after the completion of treatment, all four patients achieved complete response (CR). They are currently free from relapse and the duration of CR has been 13-72 months. During the treatment course, the level of ADAMTS13 activity and the titer of the inhibitor correlated well with resolution or exacerbation of the disease. This report suggests that rituximab exhibits short- and long-term favorable effects for the treatment of TTP and that a severe ADAMTS13 deficiency and ADAMTS13 inhibitor positivity may support early administration of rituximab in both acute/refractory and relapsing cases.