Intravenous immunoglobulin enhances the killing activity and autophagy of neutrophils isolated from immunocompromised patients against multidrug-resistant bacteria.

Intravenous immunoglobulin (IVIG) is periodically administered to immunocompromised patients together with antimicrobial agents. The evidence that supports the effectiveness of IVIG is mostly based on data from randomized clinical trials; the underlying mechanisms are poorly understood. A recent study revealed that killing of multidrug-resistant bacteria and drug-sensitive strains by neutrophils isolated from healthy donors is enhanced by an IVIG preparation. However, the effectiveness of IVIG in immunocompromised patients remains unclear. The present study found that IVIG increased both killing activity and O2(-) release by neutrophils isolated from six patients receiving immune-suppressive drugs after hematopoietic stem cell transplantation (HSCT); these neutrophils killed both multidrug-resistant extended-spectrum ß-lactamase-producing Escherichia coli (E. coli) and multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa). Moreover, IVIG increased the autophagy of the neutrophils, which is known to play an important role in innate immunity. These results suggest that IVIG promotes both the killing activity and autophagy of neutrophils isolated from immunocompromised patients against multidrug-resistant bacteria.

[Evaluation of Analytical Performance of HISCL TM, a Chemiluminescent Enzyme Immunoassay and the Use of Thrombomodulin as a Marker for Endothelial Dysfunction].

Thrombomodulin (TM) is an endothelial receptor for thrombin. The thrombin-thrombomodulin complex activates protein C in the anticoagulant pathway. Soluble TM is thought to be a marker for endothelial cell damage. We have evaluated the analytical performance of the HISCL TM test, a chemiluminescent enzyme immunoassay that measures soluble TM using biotinylated thrombomodulin monoclonal antibodies on Sysmex HISCL-2000i analyzer. Within-run coefficient of variation (CV) for control samples with low and high TM levels were 1.67% and 1.95% whereas between-run CVs for the control samples were 2.18% and 3.25% respectively. The assay showed excellent dilution linearity up to a TM level of 198 TU/mL with the lower limit of detection of 0.34 TU/mL. There was no effect of interfering substances on TM measurements. Results obtained on 362 patients showed that for those patients with a high TM level, C-reactive protein (CRP), fibrinogen (FIB), D-dimer, thrombin-antithrombin complex (TAT) and plasmin-α2 plasmin inhibitor complex (PIC) levels were significantly higher than in those patients with a normal TM level, and glomerular filtration rates (eGFR) were significantly lower than in those patients with a normal TM level. It was also observed that patients with high TM levels have significantly higher levels of von Willebrand factor antigen (vWFAg) and ristocetin cofactor activity (vWFRCo) which are associated with marked endothelial dysfunction. This study demonstrates that the HISCL TM test fulfils the analytical performance requirements for routine laboratory testing and an increased TM level detected is a useful indicator of endothelial dysfunction.

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.

PAS positivity of erythroid precursor cells is associated with a poor prognosis in newly diagnosed myelodysplastic syndrome patients.

Myelodysplastic syndrome (MDS) is a group of clonal stem cell disorders characterized by hematopoietic insufficiency. The accurate risk stratification of patients with MDS is essential for selection of appropriate therapies. We herein conducted a retrospective cohort study to examine the prognostic value of periodic acid-Schiff (PAS) reaction-positive erythroblasts in MDS patients. We examined the PAS positivity of the bone marrow erythroblasts of 144 patients newly diagnosed with MDS; 26 (18.1%) of them had PAS-positive erythroblasts, whereas 118 (81.9%) did not. The PAS-positive group showed significantly poorer karyotypes as defined in the revised International Prognostic Scoring System (IPSS-R) and higher scores in age-adjusted IPSS-R (IPSS-RA) than the PAS-negative group. Overall survival (OS) and leukemia-free survival (LFS) were also significantly shorter in the PAS-positive group than in the PAS-negative group. Similar results were obtained when only high- and very high risk groups were analyzed using IPSS-RA. This retrospective study suggested that the PAS positivity of erythroblasts is an additional prognostic factor combined with other risk scores for OS and LFS in MDS, and our results may contribute to improved clinical decision-making and rapid risk stratification.

Progressive Restrictive Ventilatory Impairment in Idiopathic Diffuse Pulmonary Ossification.

Diffuse pulmonary ossification (DPO) is a rare disease characterized by metaplastic bone formation in the lung. There are few reports with a long-term follow-up of this disease. We herein report a 47-year-old man diagnosed with idiopathic DPO at 30 years of age. The patient's vital capacity was normal until 36 years of age (3.39 L, 82.4% predicted), but it was severely decreased when he visited the hospital again at 47 years of age due to cough and dyspnea (1.98 L, 44.6% predicted). Chest computed tomography showed a significant increase in the number of high-density nodules, suggesting that the progression of DPO had caused restrictive ventilatory impairment.

Purification of leukemic blast cells from blood smears using laser microdissection.

In treatment of acute myeloid leukemia (AML), prognostic factors, including gene mutation and abnormal gene expression, enable risk stratification of patients. However, in the case of a small proportion of leukemic blast cells, such as AML associated with Down syndrome (AML-DS), it is not possible to examine prognostic factors precisely due to the large proportion of normal cells. Here, we present a novel method for examining prognostic factors by making a smear on a membrane slide glass from a small amount of diagnostic specimen and collecting highly pure leukemic blast cells by laser microdissection (LMD). We verified the effectiveness of this method using 10% KPAM1 cell line suspension and peripheral blood containing 20% blast cells obtained from a patient with transient abnormal myelopoiesis (TAM). After making blood smears, approximately 100 cells were collected and analyzed by direct sequencing. Frameshift mutations (2 bp deletion and 17 bp duplication, respectively) in GATA-1 were detected in each sample, suggesting KPAM1 and TAM blast cells were accurately purified. This novel method enables us to precisely examine prognostic factors in many cases, even in cases with a small proportion of leukemic blast cells or small specimens to preserve.

A novel variant fibrinogen, deletion of Bbeta111Ser in coiled-coil region, affecting fibrin lateral aggregation.

BACKGROUND: Functional fibrinogen concentration of a male infant showed <0.50 g/l and we speculated this patient as a dysfibrinogenemia or hypofibrinogenemia. METHODS: We analyzed propositus and his parent by DNA sequencing and by thrombin-catalyzed fibrin polymerization for purified plasma fibrinogen. RESULTS: Although functional fibrinogen determinations based on Clauss method showed the marked discrepancy of values among 3 sets of reagent and analyzer, we found a novel heterozygous variant fibrinogen, Kyoto IV, caused by 3-bp deletion in Bbeta-chain gene corresponding to the deletion of 111Ser located in coiled-coil region. We suggested that the discrepancy of fibrinogen values among 3 assays was caused by the difference in NaCl concentration in reagents for determination and analyzed the polymerization under the conditions of various NaCl concentrations. Although under normal physiological conditions Kyoto IV fibrinogen augmented the polymerization as compared with normal control, in 0.21 mol/l NaCl Kyoto IV fibrinogen showed abruptly impaired polymerization curve compared with normal control. CONCLUSION: Variant fibrinogen, BbetaDelta111Ser, showed augmented lateral aggregation under normal physiological conditions and the residue located in coiled-coil region, Bbeta111Ser, plays an important role in the lateral aggregation.

Improved detection of minimal acute myeloid leukemia cells by the use of the combined parameters of XE-2100 hematology analyzer.

BACKGROUND: For the diagnosis and therapy of acute leukemia, it is important to detect a small number of leukemic cells precisely. Although several automated hematology analyzers that carry blast-detecting programs have been developed, they do not exert sufficient detection sensitivity to exceed the sensitivity of manual eye counting method. METHODS: We constructed a new blast-detecting program by combining the numerical information acquired from five cytometric parameters presented by XE-2100. The sensitivity and specificity of this blast multi-scoring program were assessed in comparison with the Blasts flag program equipped originally in XE-2100. RESULTS: The blast-detecting sensitivity was found to be highly improved in the blast multi-scoring program as compared with the Blasts flag program without much decreasing the specificity. A small number of leukemic myeloblasts was detected at the better sensitivity than the eye counting method in the clinical course of the patients with acute myeloid leukemia. CONCLUSIONS: The daily practical use of this blast multi-scoring program will surely contribute to sensitive, objective, and real-time evaluation of the control of acute myeloid leukemia with a low cost.

Automated analysis of differentiation-induced leukemic cells during all-trans retinoic Acid therapy of acute promyelocytic leukemia.

During differentiation-induction therapy of acute promyelocytic leukemia (APL) patients with all-trans retinoic acid (ATRA), a variety of APL-derived bizarre granulocytic cells appear in the peripheral blood. To evaluate the differentiation induction of leukemic cells, we have developed a new scattergram analyzing program with an automated hematology analyzer and compared the data with the flow cytometry measuring the expression of differentiation-associated cell surface antigens, CD11b and CD16. We used the fluorescence intensity and side scatter as parameters of granulocytic maturation in the analysis with the automated hematology analyzer. The analysis of 2 ATRA-treated APL patients and in vitro study using HL-60 cells demonstrated that the levels of fluorescence intensity and side scatter decreased as accompanied with granulocytic maturation, and these changes were parallel with the results of flow cytometry. Our automated scattergram analysis of cell differentiation will contribute to general, objective, and real-time evaluation of differentiation-induction therapy of APL with ATRA.

Immature granulocyte count after liver transplantation.

We evaluated the significance of immature granulocyte (IG) count during the clinical course after liver transplantation. We counted IG using the flow cytometric method with CD16, CD11b, and CD45 antibodies. Samples were obtained from 31 patients in the Department of Transplantation and Immunology, and we determined (i) the distribution of IG peak value, (ii) the distribution of IG peak time-points, (iii) the clinical background of patients with high IG, and (iv) the clinical course of high IG cases. We observed the appearance of IG (100/microl or higher) in the majority of the patients (23 out of 31 patients; 74.2%). The IG peak was detected on the 19th day after transplantation. We observed serious complications, such as melena, rejection, or severe infection, in high IG (500/microl or higher) cases. We observed instances of inflammation with low C-reactive protein (CRP) value in the presence of IG. We believe that IG is a useful marker to monitor inflammation.