The rivaroxaban-adjusted normalized ratio: use of the prothrombin time to monitor the therapeutic effect of rivaroxaban.

Background: The prothrombin time may be used to monitor the plasma concentration of rivaroxaban. However, there is variability in the responsiveness of rivaroxaban to different thromboplastins. We aimed to develop a rivaroxaban-monitoring method using the prothrombin time to reduce the differences in the sensitivity among reagents. Methods: Rivaroxaban-spiked pooled normal plasma at a 0-1000 ng/ml concentration was used to generate a rivaroxaban-adjusted sensitivity index (SI) values, and was tested with three thromboplastins. The warfarin-adjusted international sensitivity index (ISI-warfarin), rivaroxaban-adjusted sensitivity index (SI-rivaroxaban), international normalized ratio (INR) calculated with ISI-warfarin, normalized ratio (NR) calculated with SI-rivaroxaban, and their coefficient of variances (CVs) were compared. The NR-rivaroxaban value was compared with the results of an anti-Xa assay. Results: The ISI-warfarin and SI-rivaroxaban using different thromboplastins were 1.02 and 1.88, respectively, with Thromborel S, 0.90 and 1.00 using Recombiplastin 2G, and 1.30 and 1.15 using Neoplastin CI-plus. Between-thromboplastin variability expressed as CV were 6.3%-25.1% when expressed as INR-warfarin and 1.7%-4.7% when expressed as NR-rivaroxaban. CVs for the NR-rivaroxaban with another laboratory were significantly lower than those for INR-warfarin. Anti-Xa assay v NR-rivaroxaban correlation coefficients were 0.97-0.99. Conclusion: Using a rivaroxaban-specific NR effectively minimises inter-thromboplastin variability. By utilizing a NR-rivaroxaban, standardized prothrombin time results could be rapidly obtained, especially useful in standardizing the therapeutic effect of rivaroxaban.

Validation of reflex testing rules and establishment of a new workflow for body fluid cell analysis using a Sysmex XN-550 automatic hematology analyzer.

INTRODUCTION: We developed and validated reflex testing rules for the microscopic examination (ME) of body fluids (BFs) on the Sysmex XN-550 (Sysmex Corporation) instrument. METHODS: We assessed the detection limits, precision, linearity, and carryover. To develop the reflex testing rules (derivation arm), we tested 515 samples and then validated the rules using another 507 samples (validation arm). RESULTS: All analytical performances were acceptable, and the carryover was negligible. There was agreement between the automated count and ME of red blood cells (r = .98) and total nucleated cells (TNCs) (r = .98), as well as the differential counts of neutrophils (r = .90) and lymphocytes (r = .84). We developed reflex testing rules: TNCs <10/µL, cell 2/cell 1 ratio ≤0.7, HF-BF cells >7.9/100 white blood cells, LY-X ≥85 or LY-Y ≥90, and eosinophils >2.5%. In the validation arm, implementation of the rules resulted in 126 rule-negative samples (24.9%) that were well correlated between the 2 methods. We propose a new workflow for BF cell analysis based on automated counting. CONCLUSION: The Sysmex XN-550 can be a suitable alternative to ME for BF cell analysis, especially for screening samples and subsequent automatic reporting under the rational use of laboratory-specific rules.

The added values of multiplex reverse transcriptase-PCR followed by mutation screening in the initial evaluation of acute leukemia.

INTRODUCTION: This study investigates the benefits of using multiplex reverse transcriptase-PCR (RT-PCR) in addition to standard karyotyping during the initial evaluation of acute leukemia. METHODS: A total of 1114 consecutive specimens from patients with acute leukemia were tested using a commercial multiplex RT-PCR kit (HemaVision, DNA Diagnostic). NPM1 and CEBPA mutations were selectively tested in acute myeloid leukemia (AML) patients with multiplex RT-PCR negativity. RESULTS: In specimens with optimal cytogenetics, the frequency of recurrent translocations was 31.3%, and cryptic translocations were detected in 2.1% of samples. The concordance rate between karyotyping and multiplex RT-PCR was 97.5%. In addition to the established functions, we demonstrated the additional benefits of multiplex RT-PCR, including successful molecular characterization, even in cytogenetically suboptimal specimens (5.7%); detection of submicroscopic aberrations (1.0%); detection of rare but potentially significant translocations or variants (2.5%); selection of AML candidates for mutation analysis (68.3%); and finally exclusion of recurrent translocations in patients with acute lymphoblastic leukemia or mixed phenotype acute leukemia (22.5%). CONCLUSION: We reconfirmed the accuracy and reliability of multiplex RT-PCR for diagnosing acute leukemia and demonstrated additional advantages of this system for the initial evaluation of acute leukemia. Thus, multiplex RT-PCR is worth considering in diagnostic testing of acute leukemias.

Evaluation of parameters obtained from the Sysmex XN-2000 for predicting the recovery of the absolute neutrophil count and platelets after hematopoietic stem cell transplantation.

INTRODUCTION: We analyzed abilities of parameters from Sysmex XN-2000 (Sysmex, Kobe, Japan) to predict absolute neutrophil count (ANC) and platelet recovery after hematopoietic stem cell transplantation (HSCT) in patients with hematologic malignancies. METHODS: We prospectively analyzed 911 follow-up peripheral blood samples from 44 HSCT-performed patients and evaluated the performances of the following parameters: WBC, immature granulocyte (IG), hematopoietic stem and progenitor cells (HPC), immature reticulocyte fraction (IRF), immature platelet fraction (IPF), platelet distribution width (PDW), mean platelet volume (MPV), and platelet larger cell ratio (P-LCR). RESULTS: When compared to four other parameters, the identification of initiation in IG (%)/HPC (%) increase enabled earlier prediction of ANC recovery to >500/µL and >1000/µL with more time benefit of 3.5-6.5 days/2.0-5.0 days and 3.0-6.0 days/2.0-5.0 days, respectively. When compared to IPF (%), the identification of initiation in PDW, MPV, and P-LCR (%) increase enabled earlier prediction of platelet recovery to >20 000/µL and >50 000/µL with more time benefit of 2.5-3.5 days and 2.0-3.0 days, respectively. However, the standard deviation of time benefit obtained from IG (%)/HPC (%)/PDW/MPV/P-LCR (%) was consistently large (3.0-4.3 days). CONCLUSIONS: There is a systematic pattern where a rise in most of the studied parameters can be observed in most patients before ANC/platelet recovery. However, the interindividual variation between the time of rise of these parameters and ANC/platelet recovery is large, and therefore, using these parameters to predict recovery in the individual patient is probably not meaningful in the clinical setting.

Body fluid cellular analysis using the Sysmex XN-2000 automatic hematology analyzer: focusing on malignant samples.

INTRODUCTION: The majority of previous studies on body fluid (BF) mode of automatic hematology analyzer used nonmalignant BF samples. Here, we evaluated the BF mode on the recently launched Sysmex XN for counting blood cells, especially for malignant samples. METHODS: A total of 405 BF specimens including 125 malignant samples were analyzed using both the automated method and manual microscopy. RESULTS: In non-cerebrospinal fluids (CSF) samples, there was an agreement between two methods for WBC, RBC, polymorphonuclear, and mononuclear cell counts (R(2)  = 0.96, 0.94, 0.88, and 0.88, respectively). CSF samples showed slightly poorer correlations than other fluids. Exclusion of malignant samples significantly improved correlations in non-CSF samples, but not in CSF samples. High fluorescence-BF (HF-BF) cells were identified significantly more frequently in malignant samples compared to benign samples (17.8 and 4.15/100 WBC, respectively; P < 0.001). Receiver operating characteristic curve analysis demonstrated an HF-BF cell AUC of 0.791 using a cutoff value of 6.9/100 WBC for detecting malignant samples. CONCLUSION: The BF mode on the Sysmex XN could be an alternative method for the manual counts in the BF analysis with a few drawbacks. However, if a concentration of HF-BF cells is greater than the given threshold, microscopic examination should be subsequently performed.

Sepsis affects most routine and cell population data (CPD) obtained using the Sysmex XN-2000 blood cell analyzer: neutrophil-related CPD NE-SFL and NE-WY provide useful information for detecting sepsis.

INTRODUCTION: The Sysmex XN-2000 analyzer can assess 36 routine and 57 cell population data (CPD) items. In this study, we evaluated these items as sepsis biomarkers. METHODS: We enrolled 280 normal control (NC) and 130 sepsis patients. The sepsis patients were classified as uncomplicated or complicated sepsis. Routine and CPD items were determined, and the results were compared at between the NC and sepsis groups, uncomplicated and complicated sepsis groups, and survivors and nonsurvivors. RESULTS: For the detection of sepsis, CPD items NE-SFL [defined as the fluorescent light intensity of the neutrophil area on the WDF (white blood cell differential) scattergram] and NE-WY (defined as the fluorescent light distribution width of the neutrophil area on the WDF scattergram) showed comparative or higher AUC of 0.909 and 0.905, respectively, when compared with routine items such as hematocrit, hemoglobin, RBC, RDW, immature granulocytes count, lymphocytes count, and neutrophils count. For the discrimination of sepsis severity, only platelet-related items showed higher AUC (0.723 - 0.748) than lactic acid (0.695). For the prediction of 28-day mortality, only CV and SD of RDW showed higher AUC (0.766 and 0.732 each) than lactic acid (0.712). CONCLUSIONS: Sepsis patients demonstrated significant changes in routine and CPD items related to RBC, neutrophils, lymphocytes, and platelets when compared to NCs. Increase in CPD items NE-SFL and NE-WY, which may indicate neutrophil immaturity or activation, could be useful for the detection of sepsis patients, in conjunction with currently used surrogate sepsis biomarkers. However, these items did not efficiently contribute to the discrimination of sepsis severity or predict mortality.

Radiation-induced angiogenic signaling pathway in endothelial cells obtained from normal and cancer tissue of human breast.

Despite strong possibility that endothelial cells (ECs) of tumors and normal tissues may differ in various aspects, most previous studies on ECs have used normal cells. Here, we purified ECs from tumorous and normal human breast tissues, and studied the effect of radiation on angiogenesis and relevant molecular mechanisms in these cells. We found that in normal tissue-derived ECs (NECs), 4 Gy irradiation increased tube formation, matrix metalloproteinase 2 (MMP-2) expression and extracellular signal-regulated kinase (ERK) pathway activation. In cancer-derived ECs (CECs), however, 4 Gy irradiation significantly reduced tube formation, increased the production of angiostatin and interleukin-6 (IL-6), and upregulated AKT and c-Jun N-terminal kinase (JNK) pathway activation. Knockdown experiments showed that siMMP-2 efficiently inhibited tube formation by irradiated NECs, whereas siPlasminogen effectively attenuated the radiation-induced suppression of tube formation and the upregulation of angiostatin in CECs. Moreover, siIL-6 clearly inhibited the radiation-induced generation of angiostatin in CECs. Inhibition of ERK with a pharmacological inhibitor or small interfering RNAs (siRNAs) markedly suppressed the radiation-induced tube formation and MMP-2 upregulation in NECs, whereas the inhibition of either AKT or JNK with pharmacological inhibitor or siRNA treatment of CECs markedly attenuated the inhibition of tube formation and the upregulation of angiostatin and IL-6 caused by 4 Gy irradiation. These observations collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy.

Differential diagnosis of myelofibrosis based on WHO 2008 criteria: acute panmyelosis with myelofibrosis, acute megakaryoblastic leukemia with myelofibrosis, primary myelofibrosis and myelodysplastic syndrome with myelofibrosis.

INTRODUCTION: The aim of this study was to characterize clinicopathological features of acute panmyelosis with myelofibrosis (APMF), acute megakaryoblastic leukemia with myelofibrosis (AMKL-MF), primary myelofibrosis (PMF) and myelodysplastic syndrome with myelofibrosis (MDS-MF) in order to provide the keys to the differential diagnosis of bone marrow (BM) fibrosis. METHODS: We compared age, gender, splenomegaly, serum lactate dehydrogenase level, blood cell counts, blast counts in peripheral blood (PB) and BM, megakaryocyte counts, BM cellularity, dysplasia, and the karyotypes of patients with APMF (n = 6), AMKL-MF (n = 7), PMF (n = 44), and MDS-MF (n = 44). RESULTS: APMF showed hyperplasia of all three lineages, increase in megakaryocyte count with dysplasia and frequent abnormal karyotypes. AMKL-MF was associated with elevated BM blast counts, decreased BM megakaryocyte count with rare megakaryocytic dysplasia and chromosome 21 abnormality. PMF patients displayed splenomegaly, rare blasts in PB/BM, and JAK2 V617F mutation. MDS-MF patients showed pancytopenia, dysplasia in all three lineages and recurrent chromosomal abnormalities involving chromosome 5,7,12, and 17. CONCLUSIONS: Although differential diagnosis among APMF, AMKL-MF, PMF, and MDS-MF is very challenging due to the overlapping clinical and morphological features, meticulous investigation of the patient with respect to splenomegaly, blood cell count, PB and BM findings, and karyotype will serve as a guide to correct diagnosis.

Detection of ABL1 kinase mutations in Philadelphia-positive patients exhibiting an inadequate molecular response using restriction fragment mass polymorphism and its clinical significance: a single-center experience in Korea.

INTRODUCTION: ABL1 kinase mutations represent a major mechanism of imatinib resistance in Philadelphia-positive (Ph+) patients. There is a paucity of data on ABL1 kinase mutations in Ph+ patients in Korea. METHODS: We used restriction fragment mass polymorphism (RFMP) analysis to detect ABL1 kinase mutations in blood or bone marrow specimens from 80 Ph+ patients. RESULTS: Fifty-seven patients met the criteria for inadequate molecular response (IMR). ABL1 kinase mutations were found in 2.6% of patients with chronic-phase chronic myelogenous leukemia (CML), 25.0% of accelerated-phase CML, 66.7% of blast-phase CML, and in 58.3% with Ph+ acute lymphoblastic leukemia. Twelve mutations were identified: 7 T315I, 2 E255V, 1 E255K, 1 F359V, and 1 Y253H. The majority of mutation-positive patients showed an unfavorable clinical course and often had an extra Ph or additional chromosomal abnormalities. Mutations were detected in two patients who had very low or absent BCR-ABL1 normalized ratios. CONCLUSION: Mutation analysis should be performed in Ph+ patients exhibiting an IMR to imatinib. RFMP analysis is helpful for revising therapeutic strategies because it can sensitively detect clinically relevant ABL1 kinase mutations with high frequencies.

Automated digital cell morphology identification system (CellaVision DM96) is very useful for leukocyte differentials in specimens with qualitative or quantitative abnormalities.

INTRODUCTION: The CellaVision DM96 system (CellaVision AB, Lund, Sweden) was developed as one of the automated digital cell morphology analyzer for determining leukocyte differential counts in peripheral blood smears (PBS) and we evaluated this system. METHODS: A total of 308 PB samples with abnormalities were analyzed in this study. For each sample, manual differential counts were performed by two independent technologists, and the CellaVision DM96 system was applied in duplicate. Correlations between the two methods and ability of this system to identify six abnormalities were assessed. RESULTS: The correlation coefficients between two methods were consistently high, ranged from 0.864 to 0.992. The sensitivity, specificity, positive predictive value, negative predictive values of this system for the identification of abnormalities were consistently high, especially for blasts (98.2%, 99.2%, 96.6%, 99.6%). When the instrument was ordered to count 300 or 500 cells from the operator, better performance was demonstrated than 100 cells in the leukopenic samples by sacrificing only 40 s/slide in average. CONCLUSIONS: The CellaVision DM96 system is useful in the clinical laboratory providing comparative accuracy compared with manual counts in samples with abnormalities. In leukopenic samples, report quality can be improved by ordering to count 300 or 500 cells from the operator without severe prolongation of turnaround time.

Pseudothrombocytopenia or platelet clumping as a possible cause of low platelet count in patients with viral infection: a case series from single institution focusing on hepatitis A virus infection.

INTRODUCTION: Pseudothrombocytopenia (PTCP) is the phenomenon of ethylenediaminetetraacetic acid anticoagulant-activated platelet clumping, which results in artificially low platelet counts. Other investigators have reported a few cases of PTCP associated with viral infections. The objective of this study was to demonstrate the association of viral infection with PTCP. METHODS: Medical records of patients with thrombocytopenia who were tested for peripheral blood smear examination between March 2009 and February 2011 were reviewed for platelet clumping and viral infection. RESULTS: Thrombocytopenic patients with viral infection had a higher frequency of platelet clumping than those with other diseases, which was statistically significant (13.8% vs. 6.5%, respectively: P = 0.003). Among the 18 cases where PTCP or platelet clumping was related to viral infection, hepatitis A virus infection (72.2%) was most common, followed by cytomegalovirus (11.1%) and influenza A H1N1 infections (5.6%). A third (33.3%) of the patients had platelet counts <100 × 109/L. CONCLUSION: Pseudothrombocytopenia or platelet clumping should be considered in patients with acute viral infection, particularly if the platelet count is unexpectedly low, because failure to recognize PTCP may lead to unnecessary diagnostic tests and patient mismanagement.

Direct international normalized ratio determination using multicalibrators is more responsive than the conventional method for measuring prothrombin time.

Direct international normalized ratio (INR) determination using certified INR plasmas was shown to improve precision and accuracy. We evaluated the utility of a multicalibrator in determining INR. INR values were measured in 493 blood samples from patients subjected to anticoagulation therapy (320) and control subjects (173). Study was performed using CA-7000 coagulation analyzer (Sysmex, Japan) with Thromborel S (Dade Behring, Germany). Direct INR values were obtained using PT-Multi Calibrator (Dade Behring) composed of five lyophilized calibrant plasmas. Conventional INR values were calculated from mean normal prothrombin time and instrument/reagent-specific international sensitivity index (ISI). We compared the difference between the INR results obtained with the two methods. The mean INR value of direct INR method was significantly higher than that of conventional method. The differences in values (direct INR - conventional INR) generated using the two methods increased in proportion to the INR values. Elevation of INR was observed in data obtained with the direct INR method, compared with conventional INR values. Accordingly, we conclude that direct INR method is more responsive than conventional method in determining INR.

A case of hemorrhagic gallbladder paraganglioma causing acute cholecystitis.

Gallbladder paraganglioma is a very rare tumor and so far only a few cases have been reported. Most of these were asymptomatic and were found incidentally during operation. Recently, we experienced a gallbladder paraganglioma that gave rise to hemorrhage, which in turn caused acute cholecystitis. Our case involved a 45 year-old female patient complaining of an intermittent right upper abdominal pain. After a preoperative evaluation, cholecystectomy and lymphadenectomy were performed under the impression of gallbladder cancer with acute cholecystitis. Postoperative pathologic examination revealed a hemorrhagic gallbladder paraganglioma accompanied by acute cholecystitis. Immunohistochemical staining of the chief cells for neuron specific enolase, chromogranin and synaptophysin were positive. Sustentacular cells also stained positively for S100 protein.

Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35.

The phytopathogenic bacterium Erwinia chrysanthemi (Ech) secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanases. We cloned genomic DNA from Ech PY35 digested with Sau3AI and ligated into pBluescript II SK+. One of the E. coli XL1-blue clones had the ability to hydrolyze carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning from this 2.9 kb fragment, we obtained a 2.0 kb (pPY401), designated cel5Z, which had the activity of hydrolyzation of carboxymethyl cellulose. The cel5Z gene had an open reading frame (ORF) of 1,281 bp starting with an ATG start codon and followed by a TAA stop codon, encoding 426 amino acids with a signal peptide of 41 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of CelE of Pseudomonas fluorescens, and had the conserved region, VIYEIYNEPL, it belonged to the glycoside hydrolase family 5 of EC 3.2.1.4. The molecular mass of Cel5Z protein from E. coli XL1-blue, as analyzed by CMC-SDS-PAGE, appeared to be 42 kDa. The optimum pH was 6, and the optimum temperature was about 40 degrees C for its enzymatic activity.

Characterization of Erwinia chrysanthemi PY35 cel and pel gene existing in tandem and rapid identification of their gene products.

Genomic DNA of the phytopathogenic Erwinia chrysanthemi PY35 was partially digested with Sau3AI, ligated into the BamHI site of pBluescript II SK+, and introduced into E. coli. One clone that was able to hydrolyse carboxymethylcellulose and polygalacturonic acid was selected. A 2.9 kb fragment containing the pelL1 gene (pPY300) and cel5Z gene (pPY401) in tandem was subcloned and sequenced. The pelL1 and cel5Z genes had open reading frames of 1,278 bp and 1,281 bp encoding 425 and 426 amino acid residues with calculated molecular weights of 45,649 Da and 46,473 Da, respectively. pelL1 and cel5Z carried a typical prokaryotic signal peptide of 24 and 41 amino acid residues, respectively. The apparent molecular masses of the proteins when expressed in E. coli cells were approximately 43 kDa (PelL1) and 42 kDa (Cel5Z) as assessed by PGA-SDS-PAGE and CMC-SDS-PAGE.

Volvulus of the splenic flexure of the colon.

The definition of volvulus is an axial twist of a portion of the gastrointestinal tract along its mesentery. The involved bowel is obstructed partially or completely with a variable degree of arterial and venous occlusion. The colon is the most common site for volvulus. The splenic flexure is the least common site of colonic volvulus. We experienced a case of the volvulus of the splenic flexure. It will be the 30th case of the volvulus involving the splenic flexure in the English literature, to our knowledge. A 30-year-old woman was admitted due to abdominal pain and distention with vomiting. An emergency barium study revealed characteristic "bird beak" sign. Surgery was performed resecting the involved colon of splenic flexure. The result was excellent.