The value of the portable fibrinogen measuring device-a case report of severe postpartum hemorrhage with obstetric disseminated intravascular coagulation.

BACKGROUND: Fibrinogen concentration is an important indicator of the treatment for obstetric disseminated intravascular coagulation (DIC). We present how using the fibrinogen measuring device could solve problems in the treatment of postpartum hemorrhage with complicated DIC. CASE PRESENTATION: A 32-year-old woman with monochorionic diamniotic twins at 22 weeks of pregnancy was diagnosed with placental abruption and underwent emergent cesarean section. The estimated blood loss was 8375 g. She was transferred to our hospital for further treatment. Compressive uterine sutures and balloon tamponade were performed. We transfused fibrinogen and fresh frozen plasma actively during the operation to maintain plasma fibrinogen above 200 mg/dL by using a point-of-care fibrinogen measuring device. In spite of massive hemorrhage exceeding 10 L, she was extubated at the end of the operation and discharged on the 7th day after the operation. CONCLUSION: The portable fibrinogen measuring device was useful for point-of-care assessment of obstetric DIC.

Soluble Interleukin-2 Receptor Index Predicts Outcomes After Cord Blood Transplantation.

BACKGROUND: Our previous study demonstrated that the soluble interleukin-2 receptor (sIL-2R) index, defined as the ratio of serum sIL-2R levels at neutrophil engraftment to that before conditioning, is a biomarker that can predict acute graft-vs-host disease (GVHD) after unrelated bone marrow transplantation. In the present study, we evaluated the significance of the sIL-2R index among patients who underwent cord blood transplantation (CBT). METHODS: We retrospectively analyzed 31 patients who underwent single-unit CBT as their first transplantation for hematologic malignancies. RESULTS: The median sIL-2R index was 4.2. The cumulative incidence of grade II to IV acute GVHD was not associated with the sIL-2R index. However, the cumulative incidence of relapse at 3 years after transplantation was significantly lower, with an sIL-2R index ≥ 3.7 than with an index < 3.7 (12.8% vs 50.0%; P = .04). As a result, the probability of overall survival at 3 years after transplantation was significantly higher in the former group than in the latter (79.8% vs 20.0%; P < .01). Only the dose of corticosteroid administered in the pre-engraftment period influenced the sIL-2 index. CONCLUSION: The sIL-2R index can predict the incidence of relapse and probability of survival after CBT, possibly reflecting a graft-vs-leukemia effect.

TERT and JAK2 polymorphisms define genetic predisposition to myeloproliferative neoplasms in Japanese patients.

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are often characterized by specific somatic mutations in any of the three genes: JAK2, CALR, or MPL. A single nucleotide polymorphism (SNP), rs2736100, in the reverse transcriptase gene (TERT) and a germline JAK2 46/1 haplotype have been associated with MPNs in North American and European patients. We examined 201 Japanese MPN patients, including 52 with PV, 131 with ET, and 18 with PMF, as well as 366 control individuals for TERT rs2736100 and JAK2 rs10974944, a tagging SNP of the 46/1 haplotype. Furthermore, correlations between the JAK2 V617F allele burden at diagnosis and TERT rs2736100 or JAK2 rs10974944 were evaluated using a digital PCR assay for accurate quantitation. The JAK2 46/1 haplotype, but not the TERT rs2736100 SNP, was correlated to the JAK2 V617F mutant allele burden in JAK2 V617F-positive MPN patients. In conclusion, we demonstrated that both TERT rs2736100_C and JAK2 46/1 haplotype are predisposing factors for MPNs in Japanese patients. While TERT rs2736100_C tended to have a more general, non-specific effect on all MPNs, the JAK2 46/1 haplotype was essentially predisposed to the JAK2 V617F-positive MPNs.

[Refractory primary immune thrombocytopenia in pregnancy requiring splenectomy and repeated intravenous immunoglobulin therapy].

A 30-year-old primigravid woman without a history of thrombocytopenia was referred to our hospital because of severe thrombocytopenia (<1,000 thrombocytes/µl) at 16 weeks of gestation and diagnosed with idiopathic thrombocytopenic purpura (ITP). There was no improvement in the platelet count after treatment with 0.5-1.0 mg/kg/day prednisolone, and the 400 mg/day intravenous immunoglobulin (IVIg) administered for 5 days gradually became ineffective; therefore, a laparoscopic splenectomy was performed at 25 weeks of gestation. The increase in the platelet count after the splenectomy was temporary, but the effects of IVIg therapy improved, and the patient received IVIg therapy seven times in total during her pregnancy. Her platelet count ranged between 10,000 and 70,000/µl after the splenectomy, compared with <5,000/µl before the surgery. The patient underwent an elective cesarean section at 34 weeks of gestation without any significant bleeding. The baby was diagnosed with thrombocytopenia at birth (34,000 thrombocytes/µl) and was administered only one dose of IVIg, which increased the baby's platelet count to a normal level after 14 days. The patient's platelet count also increased after delivery. Splenectomy and repeated IVIg therapy can be considered for refractory severe ITP during pregnancy.

Successful Treatment of Behçet's Disease Associated with Acute Myeloid Leukemia with Myelodysplasia-related Changes Using Azacitidine and Tacrolimus before Allogeneic Hematopoietic Stem Cell Transplantation.

The coexistence of acute myeloid leukemia (AML) with Behçet's disease (BD) is rare. The optimum treatment for AML-associated BD has not been established. We herein report a patient with BD who developed AML with myelodysplasia-related changes. Induction chemotherapy caused complete remission of the AML but worsened the BD. Thereafter, AML was treated with azacitidine. The BD was steroid-dependent. Tacrolimus was added, which improved the BD. The patient underwent allogeneic hematopoietic stem cell transplantation (HSCT) and remains in complete remission for both diseases. Allogeneic HSCT was found to be a potent therapeutic option for AML-associated BD. In addition, azacitidine and tacrolimus were shown to be a suitable bridging regimen before HSCT.

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

Soluble interleukin-2 receptor index predicts the development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation from unrelated donors.

Acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) is associated with significant morbidity and mortality. In the present study, we retrospectively evaluated whether soluble interleukin-2 receptor (sIL-2R) index, defined as the ratio of serum sIL-2R levels at neutrophil engraftment to those at the pre-conditioning regimen, was predictive of acute GVHD among 51 patients who underwent allogeneic HSCT as their first transplantation and achieved engraftment. The median sIL-2R index was 3.6, and the sIL-2R values were positively associated with acute GVHD severity (grade 0-I: 3.8 ± 2.0 vs. grade II-IV: 7.1 ± 5.7; P = 0.05). Grade II-IV acute GVHD had a cumulative incidence of 31.4 %, and was significantly more frequent among patients with an sIL-2R index of ≥4.5 (≥4.5: 50.0 % vs. <4.5: 21.2 %; P = 0.03). Multivariate analysis revealed that an sIL-2R index of ≥4.5 [hazard ratio (HR) 3.5, P < 0.01] and donor age of >35 years (HR 3.8, P = 0.02) were significant risk factors for grade II-IV acute GVHD. Therefore, increased sIL-2R levels from baseline to engraftment might predict the risk of moderate-to-severe acute GVHD after allogeneic HSCT from an unrelated donor.

A Unique Primer with an Inosine Chain at the 5'-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method.

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3'-terminus of each primer. To use this method at least two allele-specific primers and one "counter-primer", which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3'-terminus, and another primer should have a few non-complementary flaps at the 5'-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5'-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method.

Altered expression of circadian clock genes during peripheral blood stem cell mobilization induced by granulocyte colony-stimulating factor.

Circulating hematopoietic stem cells exhibit robust circadian fluctuations, which influence the mobilized cell yield, even during enforced stem cell mobilization. However, alterations in the expression of circadian clock genes during granulocyte colony-stimulating factor (G-CSF)-induced peripheral blood stem cell (PBSC) mobilization are not fully elucidated. Therefore, we measured the expression of these genes in human peripheral blood leukocytes from 21 healthy donors. While CRY1 mRNA expression significantly increased by 3.9-fold (p < 0.01), the expression of PER3, CRY2 and BMAL1 mRNAs significantly decreased (by 0.2-fold, 0.2-fold, and 0.6-fold, respectively; p < 0.001) after G-CSF administration. Moreover, CRY1 mRNA expression was inversely correlated with the plasma level of noradrenaline (r = -0.36, p < 0.05), while PER3, CRY2, and BMAL1 mRNA expression directly correlated with the plasma level of noradrenaline (r = 0.55, r = 0.66, and r = 0.57, respectively; p < 0.001). Thus, significant correlations between the levels of circadian clock gene mRNAs and the plasma level of noradrenaline, a sympathetic nervous system neurotransmitter, were established. The modulation of sympathetic activation and of the circadian clock may be novel therapeutic targets for increasing stem cell yields in PBSC donors.

JAK2 46/1 haplotype is associated with JAK2 V617F-positive myeloproliferative neoplasms in Japanese patients.

Myeloproliferative neoplasms (MPNs) constitute a group of phenotypically diverse chronic myeloid malignancies, characterized by clonal hematopoiesis and excessive production of terminally differentiated myeloid blood cells. The MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), most of which are characterized by a somatic point mutation, V617F, in the janus kinase 2 (JAK2) gene. This mutation was recently shown to occur more frequently in a specific JAK2 haplotype, JAK2 46/1, in North American and European MPN patients. Little is known, however, about JAK2 haplotypes in Japanese MPN patients. Therefore, we examined 108 Japanese patients with MPN, including 19 with PV, 61 with ET, 10 with PMF, and 17 with unclassifiable MPN, as well as 104 control individuals for the JAK2 rs10974944(C/G) single nucleotide polymorphism, in which the G allele indicates the 46/1 haplotype. We found that the JAK2 46/1 haplotype was significantly more frequent in patients with V617F-positive MPN than in controls (odds ratio [OR], 3.6; 95 % confidence interval [CI], 2.2-5.8, p < 0.001), and in PV patients than in controls (OR, 6.3; 95 % CI, 3.0-29.4, p < 0.001). In conclusion, we demonstrated that the JAK2 46/1 haplotype is associated with JAK2 V617F-positive MPNs in Japanese patients.

Sequential injection immunoassay for environmental measurements.

Sequential injection immunoassay systems for environmental measurements based on the selective immunoreaction between antigen and antibody were described. A sequential injection analysis (SIA) technique is suitable to be applied for the procedure of enzyme-linked immunosorbent assay (ELISA), because the washing and the addition of reagent solutions can be automated by using a computer-controlled syringe pump and switching valve. We selected vitellogenin (Vg), which is a biomarker for evaluating environmental risk caused by endocrine-disrupting chemicals in the hydrosphere, and linear alkylbenzene sulfonates (LAS) and alkylphenol polyethoxylates (APEO), which are versatile surfactants, as target analytes in the flow immunoassay systems. For Vg monitoring, SIA systems based on spectrophotometric, chemiluminescence, and electrochemical determinations were constructed. On the other hand, chemiluminescence determination was applied to the detection of LAS and APEO. For APEO, an SIA system combined with surface plasmon resonance (SPR) sensor was also developed. These new sequential injection immunoassay systems are expected to be useful systems for environmental analysis.

Hematopoietic progenitor cell count, but not immature platelet fraction value, predicts successful harvest of autologous peripheral blood stem cells.

Mobilized stem cells in the peripheral blood (PB) must be efficiently harvested at the appropriate time before autologous PB stem cell (PBSC) transplantation. Enumeration of CD34+ cells in the PB before apheresis predicts the number of PBSCs that can be collected, but the cytometric techniques used are complex and expensive. Therefore, it is necessary to identify an alternative to the CD34+ cell count in PBSC harvest-time monitoring. Fully automated flow cytometry using blood cell counters now allows reliable quantification of immature myeloid cells in the PB, referred to as hematopoietic progenitor cells (HPC), and reticulated platelets, expressed as the immature platelet fraction (IPF). Immature or reticulated platelets are thought to correlate with thrombopoietic activity of the marrow. Following a chemotherapy nadir, the recovery of white blood cell and platelet counts has been used to determine the right time for apheresis. Therefore, we examined whether the HPC count and IPF value could be used to predict PBSC mobilization in 20 patients with hematological malignancies. The HPC count was found to be correlated with the CD34+ cell count (r = 0.84, P < 0.01), whereas the IPF value was not (r = 0.37, P = 0.44). Therefore, the HPC count, but not the IPF value, is a possible predictor of the timing of autologous stem cell transplantation.

Cultivated grapevines represent a symptomless reservoir for the transmission of hop stunt viroid to hop crops: 15 years of evolutionary analysis.

Hop stunt was a mysterious disorder that first emerged in the 1940s in commercial hops in Japan. To investigate the origin of this disorder, we infected hops with natural Hop stunt viroid (HpSVd) isolates derived from four host species (hop, grapevine, plum and citrus), which except for hop represent possible sources of the ancestral viroid. These plants were maintained for 15 years, then analyzed the HpSVd variants present. Here we show that the variant originally found in cultivated grapevines gave rise to various combinations of mutations at positions 25, 26, 54, 193, and 281. However, upon prolonged infection, these variants underwent convergent evolution resulting in a limited number of adapted mutants. Some of them showed nucleotide sequences identical to those currently responsible for hop stunt epidemics in commercial hops in Japan, China, and the United States. Therefore, these results indicate that we have successfully reproduced the original process by which a natural HpSVd variant naturally introduced into cultivated hops was able to mutate into the HpSVd variants that are currently present in commercial hops. Furthermore, and importantly, we have identified cultivated grapevines as a symptomless reservoir in which HSVd can evolve and be transmitted to hop crops to cause epidemics.