Takashi Horii

Association between antimicrobial consumption and clinical isolates of methicillin-resistant Staphylococcus aureus: a 14-year study

The objective of this study was to determine the relationship between clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and antimicrobial consumption in hospitalized patients over a 14-year period. The study was retrospectively conducted between January 1995 and December 2008 at Juntendo University Hospital, Tokyo, Japan, a 1,020-bed tertiary-care teaching hospital. The incidence of MRSA isolates was examined using clinical specimens presented to the microbiology laboratory in the hospital. Antimicrobial consumption through intravenous injection was calculated in terms of the number of defined daily doses per 100 bed-days. The correlation between the incidence of MRSA isolates and antimicrobial consumption was determined employing a multiple stepwise regression analysis. A total of 109,946 bacterial isolates were consecutively collected over the 14-year period, and, of these, 13,872 (64% of S. aureus strains excluding coagulase-negative staphylococci) were MRSA strains. The longitudinal observation showed that the number and rate of MRSA isolates marginally decreased. The rate of MRSA isolates among S. aureus strains in 1995 was 68.5%, whereas that in 2008 was 53.8%. Consumption of cephalosporins decreased. Among carbapenems, the rate of imipenem (IPM) consumption decreased, whereas that of meropenem increased. A multiple stepwise regression analysis revealed that the antimicrobial consumption of cefmetazole, cefotiam, and IPM was positively correlated with the incidence of MRSA isolates. The use of β-lactam antimicrobials may contribute to the development of MRSA strains.

The use of CellaVision competency software for external quality assessment and continuing professional development

Aims Quality assessment of blood cell morphological testing, such as white blood cell (WBC) differential and its interpretation, is one of the most important and difficult assignments in haematology laboratories. A monthly survey was performed to assess the possible role of the proficiency testing program produced by CellaVision competency software (CCS) in external quality assessment (EQA) of the clinical laboratories of affiliated university hospitals and the effective utilisation of this program in continuing professional development (CPD). Methods Four monthly proficiency surveys were conducted in collaboration with four clinical laboratories affiliated with the teaching hospitals of Juntendo University of Medicine in Japan. Results EQA results by the CCS proficiency testing program revealed a difference of performance levels of WBC differential and morphological interpretation and a discrepancy in the WBC differential criteria among laboratories. With regard to the utilisation of this proficiency program as a tool for CPD, this program successfully improved the performance of the low-scoring laboratories and less experienced individuals. Conclusions The CCS proficiency testing program was useful for the quality assessment of laboratory performance, for education, and for the storage and distribution of cell images to be utilised for further standardisation and education.

Performance evaluation of the digital cell imaging analyzer DI-60 integrated into the fully automated Sysmex XN hematology analyzer system

Abstract Background: The XN-Series (Sysmex, Kobe, Japan) have been equipped with the automated digital cell imaging analyzer DI-60, which provides complete automation of the sample processing with automated complete blood counts (CBC), slide making/staining, and digital scanning with cell pre-classification. The aim of this study was to evaluate the efficacy of the XN-Series as an integrated blood cell analysis system. Methods: White blood cell (WBC) morphological analysis by the DI-60 was evaluated using 232 blood samples from patients. Routine analysis of a total of 2000 blood samples has been performed to evaluate the processing ability of the XN-Series connected to the DI-60. Results: The overall analysis accuracy of pre-classification of WBC by the DI-60 was 88.4%. Good correlation was observed between final results of the DI-60 analysis and manual differentiation with high sensitivity and specificity for blasts and immature granulocytes. The sample processing time of the XN-Series, from automated CBC to cell pre-classification, was 38±1 min/single run and 165±12 min/500 CBC samples run (slide preparation rate 15.6%) with no sample hold-up at the DI-60. Conclusions: The automated morphological analysis capability of the DI-60 has potential usefulness in the integrated automated hematology analysis system of XN-Series.

Harmonization of quantitative BCR-ABL measurements using the secondary reference material anchored to the WHO primary standards.

Sir, The oncogenic BCR-ABL fusion gene is a hallmark of chronic myeloid leukemia (CML), and tyrosine kinase inhibitors (TKIs) specifically disable the constitutive activity of the BCR-ABL kinase [1–4]. Responses to TKI treatment were defined using the practice guidelines issued by the National Comprehensive Cancer Network (NCCN) and the European Leukemia Net (ELN), which recommend measuring BCR-ABL gene expression relative to that of a control gene in bone marrow or peripheral blood samples [5–7]. The International Randomized Study of Interferon and STI571 (IRIS) demonstrated that a 3-log reduction of BCR-ABL mRNA compared with a standardized transcript baseline level of diagnostic CML is a desirable therapeutic response [1, 8]. Because of an extensive variation in the BCR-ABL mRNA levels reported by individual laboratories [9, 10], efforts have been made to standardize the reporting of real-time quantitative polymerase chain reaction (RQ-PCR) results using the International Scale (IS) percentage. The IS percentage (% BCR-ABL IS) does not require a baseline value at diagnosis, and it exhibits better correlation with the complete cytogenetic response (CCyR) than the log reduction method [11]. A sample exchange system with reference laboratories has been shown to be effective in comparing the results from different laboratories. However, the validation procedure is a lengthy and costly and requires continuous revalidation [12–14]. Therefore, now, the effort is ongoing to distribute IS value predefined reference materials. The World Health Organization International Genetic Reference Panel for the quantitation of BCR-ABL1 mRNA (World Health Organization document, World Health Organization/BS/09.2106) has been distributed to manufacturers to generate secondary reference materials [13]. Currently, several manufacturers are attempting commercial production of secondary materials. It was recently reported that a synthetic Armored RNA Quant IS Calibrator Panel (ARQ IS; Asuragen, Inc., Austin, TX, USA) provides accurate BCRABL IS values [14]. In this study, we aimed to utilize commercially produced secondary reference materials to standardize BCR-ABL measurements. We calibrated our laboratory-developed (local) RQ-PCR values using synthetic ARQ IS reference panels with a sensitivity of 0.0033% of the IS. Peripheral blood samples were collected from 38 CML patients who achieved a complete hematological response (defined as normalization of blood cell counts with no immature cells, <5% basophils and no palpable spleen) [15] receiving imatinib, nilotinib, dasatinib, or interferon therapy at the Saga University Hospital in Japan between February and April 2013. The diagnosis of CML was determined through the detection of the Philadelphia chromosome using cytogenetic evaluation and/or BCR-ABL transcription using RQ-PCR. Patients provided written informed consent, and the study was approved by the local research ethics committee. Total leukocyte RNA was extracted using a QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany), and Transcriptor Universal cDNA Master reverse transcriptase (Roche Diagnostics, Mannheim, Germany) was used for cDNA synthesis. cDNA was amplified by 55 cycles of RTPCR using the LightCycler 2.0 (Roche Diagnostics) and LightCycler TaqMan Master in accordance with the manufacturer’s instructions. ABL was used as the control gene. The primers and the probes used were as follows: BCR-ABL; forward primer, 50-TGACCAACTCGTGTGTGA AACTC-30; reverse primer, 50-CACTCAGACCCTGAGGCT CAA-30; probe, 50-CCCTTCAGCGGCCAGTAGCATCTGA -30, ABL; forward primer, 50-CGAAGGGAGGGTGTACCAT TA-30; reverse primer, 50-CAACTCGGCCAGGGTGTT-30; probe, 50-CTTCTGATGGCAAGCTCTACGTCTCCTCC-30. The sequences were obtained from GenBank Accession No. X02596 for BCR and No. X16416 for ABL. The probe contained the fluorescent reporter dye 6-carboxyfluorescein (FAM) at the 50-end and the fluorescent quencher dye Black Hole Quencher (BHQ) at the 30-end. The results were reported as a BCR-ABL/ABL ratio (%). An in vitro transcribed RNA of the BCR-ABL gene from the K562 cell line was used to determine the lower

Automated mixing studies and pattern recognition for the laboratory diagnosis of a prolonged activated partial thromboplastin time using an automated coagulation analyzer

INTRODUCTION The objective of this study was to explore whether an automated coagulation analyzer could be applied to normal plasma mixing studies for the assessment of blood samples showing a prolonged activated partial thromboplastin time (APTT). MATERIALS AND METHODS Ten laboratory staff members performed normal plasma mixing studies and evaluated plasma samples using 3 different methods: (1) manual dilution and analysis, (2) manual dilution and automatic analysis with STA-R®, and (3) automatic dilution and analysis with the Coapresta® 2000 (CP2000). The time from the start of the analysis to the generation of the result plots and the plasma volumes required were determined. We analyzed patient plasma samples showing a prolonged APTT using the CP2000, and the result plots were categorized into 3 curve patterns based on the area ratio values: the inhibitor type (convex pattern), deficiency type (concave pattern), and suspicious inhibitor type (approximately straight pattern). RESULTS When pooled patient plasma was used, the same patterns were obtained from normal plasma mixing studies using the 3 different methods. The time required to complete the mixing studies and the plasma volumes required were 28.2 ± 2.4 min and 350 μL for manual analysis, 23.2 ± 2.1 min and 875 μL for STA-R(®), and 8.5 +/- 0.1 min and 175 μL for CP2000, respectively. Of 31 patient samples, 9 were categorized into the inhibitor type, 15 were categorized into the deficiency type, and 7 were categorized into the suspicious inhibitor type. CONCLUSIONS The CP2000 analyzer is applicable to the laboratory diagnosis of a prolonged APTT using pattern recognition, as it requires a shorter time to complete mixing studies and a smaller plasma volume in comparison with manual analysis.

Evaluation of Factor Xa-Specific Chromogenic Substrate Assays and the Determination of Pharmacokinetics of Fondaparinux

Fondaparinux (FPX), a synthesized factor Xa inhibitor, is one of the most popular anticoagulants for the prevention of postoperative venous thromboembolism (VTE). Although routine monitoring is not required, the bleeding adverse events cannot be neglected, and the measurement of anti-Xa activity is expected to be monitored. The primary purpose of this study is to evaluate the performances of 2 chromogenic assays for the detection of anti-Xa activity. Furthermore, the pharmacokinetics of FPX was examined using chromogenic assays. Anti-Xa activity was measured using 2 FPX-based chromogenic substrates (S2222 and STA-Liquid Anti-Xa). The reproducibility, detection limits, linearity, and correlations between the substrates were examined using normal plasma doped with low and high concentrations of FPX formulation. In addition, anti-Xa activity in 235 clinical samples from 164 cases treated was measured, and the pharmacokinetics of FPX was evaluated. Both of the tested substrates were capable of accurately measuring the anti-Xa activity of FPX, with a lower limit of 0.05 μg/mL and a coefficient of variation of less than 10%. The repeated administration of FPX induced a gradual but significant increase in the anti-Xa activity, which was negatively correlated with body weight and estimated glomerular filtration rate. No significant correlation between the anti-Xa activity and the occurrence of postoperative VTE or bleeding event was observed. Anti-Xa activity can be successfully determined using 2 chromogenic assays and automated biochemical analyzers. The clinical significance of anti-Xa activity monitoring should be examined in the future study.

The efficacy of an internet-based e-learning system using the CellaVision Competency Software for continuing professional development.

*Corresponding author: Yoko Tabe, Department of Laboratory Medicine, Juntendo University of Medicine, 2-1-1 Hongo , Bunkyoku, Tokyo 113-8421, Japan, E-mail: tabe@juntendo.ac.jp Yuki Horiuchi, Mayumi Idei and Takashi Miida: Department of Laboratory Medicine, Juntendo University of Medicine, Tokyo, Japan Kanako Kasuga: CellaVision Japan, Hokohama, Japan Imiko Maenou and Takashi Horii: Clinical Laboratory, Juntendo University Hospital, Tokyo, Japan Akimichi Ohsaka: Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University School of Medicine, Tokyo, Japan Letter to the Editor

Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called “the Eprobe leukemia assay,” for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

Evaluation of cell count and classification capabilities in body fluids using a fully automated Sysmex XN equipped with high-sensitive Analysis (hsA) mode and DI-60 hematology analyzer system

The XN series automated hematology analyzer has been equipped with a body fluid (BF) mode to count and differentiate leukocytes in BF samples including cerebrospinal fluid (CSF). However, its diagnostic accuracy is not reliable for CSF samples with low cell concentration at the border between normal and pathologic level. To overcome this limitation, a new flow cytometry-based technology, termed “high sensitive analysis (hsA) mode,” has been developed. In addition, the XN series analyzer has been equipped with the automated digital cell imaging analyzer DI-60 to classify cell morphology including normal leukocytes differential and abnormal malignant cells detection. Using various BF samples, we evaluated the performance of the XN-hsA mode and DI-60 compared to manual microscopic examination. The reproducibility of the XN-hsA mode showed good results in samples with low cell densities (coefficient of variation; % CV: 7.8% for 6 cells/μL). The linearity of the XN-hsA mode was established up to 938 cells/μL. The cell number obtained using the XN-hsA mode correlated highly with the corresponding microscopic examination. Good correlation was also observed between the DI-60 analyses and manual microscopic classification for all leukocyte types, except monocytes. In conclusion, the combined use of cell counting with the XN-hsA mode and automated morphological analyses using the DI-60 mode is potentially useful for the automated analysis of BF cells.

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode “XN-BF gating algorithm” to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples.