A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

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.

Efficacy of PCR-based open reading frame typing assay for outbreak investigation of metallo-ß-lactamase-producing Pseudomonas aeruginosa in hematology unit.

We investigated the efficacy of the PCR-based open reading frame typing (POT) assay for outbreak investigation of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa (MBL-PA). A total of 53 P. aeruginosa isolates were detected between January 2010 and December 2012 on a hematology ward, of which 6 were identified as MBL-PA with the blaIMP-1 gene. The POT assay revealed the same genotype (207-41) in 3 of 6 MBL-PA, suggesting an outbreak caused by a single strain. Environmental investigation of bathroom samples revealed the same POT genotype (207-41) as those of the clinical isolates and no other MBL-PA strains. Genetic relatedness of the MBL-PA isolates was confirmed by the DiversiLab repetitive-sequence-based PCR typing system, suggesting the POT type 207-41 as a genetically identical clone. The POT assay can be successfully applied to MBL-PA genotyping.

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.

Comparison of the Analytical Performance Between cobas EGFR Assay and PCR-Clamp Method in the Detection of EGFR Mutations in Japanese Non-Small Cell Lung Cancer Patients.

BACKGROUND: EGFR, a tyrosine-kinase, plays an important role in the progression of lung cancer. Since genetic abnormality of EGFR alters the effects of tyrosine-kinase inhibitors targeting EGFR, molecular analyses of EGFR have recently gained more attention in the treatment of lung cancer. However, several different techniques are available and which method is superior has not been determined. In this study, we compared two recently developed PCR-based techniques, PCR-clamp method and cobas EGFR assay. METHODS: Ninety-four surgical samples and 58 biopsy samples from patients suffering from non-small cell lung cancers (NSCLCs) were included in the study. Samples with positive and negative genetic abnormalities, 66 and 28 respectively, were chosen for PCR-Clamp methods. Those same samples were reanalyzed with cobas EGFR assay. RESULTS: The concordance between PCR-Clamp and cobas EGFR methods was 95.7%. PCR-Clamp failed to detect four mutations that were detected with cobas EGFR assay. These two methods were further tested by analyzing 58 random biopsy samples. The concordance for the biopsy samples was 93.1%, and PCR-Clamp, again, failed to detect three mutations that were detected with cobas EGFR assay. CONCLUSIONS: Our results showed both methods detected most of the known EGFR mutations and the concordance was similar to those previously reported in different ethnicities. However, in our study, PCR-Clamp method failed to detect a total of seven mutations that were detected with cobas EGFR assay. Thus, we concluded that cobas EGFR assay is an easier and more accurate screening assay than PCR-Clamp method in detecting EGFR genetic abnormalities.

Pancreatic lipase activity in overnight effluent predicts high transport status in peritoneal dialysis patients.

BACKGROUND: Long-term peritoneal dialysis (PD) causes peritoneal morphological and functional changes, resulting in high transport status featuring increased peritoneal permeability. High transport status is diagnosed by peritoneal equilibration test (PET), a reliable but time-consuming method. We identifed a reliable biomarker in peritoneal effluent to predict high transport status in PD patients. METHODS: We collected peritoneal effluent and serum from 33 PD patients and measured common laboratory test parameters. High transport status was determined by PET if the dialysate/plasma ratio of creatinine at 4h dwell (D/P Cr 4h) was ≥0.81. RESULTS: There were significant correlations between D/P Cr 4h and some laboratory parameters in overnight effluent (pancreatic lipase activity, r=0.65, p<0.001; ß2-microglobulin concentration, r=0.59, p<0.001; IL-6 concentration, r=0.53, p<0.001; and CA125 concentration, r=0.29, p=0.027). In a multivariate logistic regression analysis, the pancreatic lipase activity in overnight effluent was identified as an independent predictor of high transport status even after adjusting for age, PD duration, and glomerular filtration rate [OR=1.43 (95% CI: 1.11-1.83), p=0.005]. CONCLUSIONS: The pancreatic lipase activity in overnight effluent is an independent predictor of high transport status in PD patients.

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.

Clinical Performance Evaluation of a High-Risk Human Papillomavirus Genotyping Test "Clinichip HPV" Using Cervical Scrape Specimens.

BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) is closely associated with cervical cancer development. In this study, the performance of the Clinichip HPV genotyping assay as a screening laboratory test for high-risk HPV infection was evaluated. METHODS: The genotypes of 74 cervical scrape specimens were tested using the Clinichip HPV assay and a conventionally employed HPV polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. PCR sequencing was performed in cases with discrepant results between the Clinichip HPV test and PCR-RFLP. RESULTS: Genotyping using the Clinichip HPV assay and PCR-RFLP method resulted in 27% disagreement. PCR sequence results exhibited 79% and 21% consistency with the Clinichip HPV assay and PCR-RFLP method, respectively. Multiple infections were detected in 24.3% and 12.2% of the tested cases using the Clinichip HPV assay and PCR-RFLP method, respectively. CONCLUSIONS: The genotyping performance of the Clinichip HPV showed strong concordance with PCR sequencing, although this rate was partially diminished in cases with multiple HPV infections. The Clinichip HPV represents a suitable laboratory test for the clinical screening of high-risk HPV infections.

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

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.

Human mesenchymal stem cells differentiate to epithelial cells when cultured on thick collagen gel.

The stem cell niche is crucial to the control of stem cell fate determination in vitro as well as in vivo, and an understanding of these niches is required for the progression of stem cell and tissue engineering. The goal of our study was to commit human mesenchymal stem cells (hMSCs) to the epithelial lineage. To do this, we cultured bone marrow-derived mesenchymal stem cells (MSCs) on plates coated with type I collagen gel with or without 10 µM all-trans retinoic acid (ATRA).We found depth-dependent differentiation of hMSCs to the epithelial lineage, with the thick collagen gel (1900 µm) generating more than 80% cytokeratin-18 (CK-18)-positive cells, whereas the thin collagen gel (100 µm) generated significantly fewer CK-18-positive cells. In addition, we found that supplementation of 10 µM ATRA enhanced CK-18 expression and induced cluster-formation in cells grown on the thick collagen gel. The effect of gel depth on hMSC differentiation appears to be caused by partial cytoskeletal disruption.These results suggest that ATRA and a collagen extracellular matrix may have a synergistic effect on differentiation of human mesenchymal stem cells to epithelial lineage.

Performance evaluation of the automated morphological analysis of erythrocytes by CellaVision DM96.

BACKGROUND: Automated digital morphology systems are utilized for blood cell morphological examination. The aim of this study is to evaluate the accuracy and efficacy of RBC morphological anomaly screening using the CellaVision DM96 (DM96) automated image analysis system. METHODS: The automated analysis of RBC shape, size, and chromasia abnormalities was conducted on the DM96 using 478 blood samples. A manual microscopic review was independently performed. RESULTS: The DM96 preclassified samples as poikilocytosis-positive for 98% of cases with schistocytosis or echinocytosis, 97% of elliptocytosis, and 92% or 65% of cases that were positive for teardrop cells or for target cells, respectively. The accuracy of the DM96 in the detection of RBC size and chromasia abnormalities of iron deficiency anemia cases was higher than direct microscopic observation. CONCLUSIONS: Automated morphological analysis with the DM96 has potential utility in the morphological screening of RBC anomalies that are associated with disease.

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 ß-lactam antimicrobials may contribute to the development of MRSA strains.

Regulation of osteogenetic differentiation of mesenchymal stem cells by two axial rotational culture.

It is crucial to understand how gravitational force affects the osteogenic differentiation of mesenchymal stem cells (MSCs), and these fundamental aspects hold promise for the development of a novel model of MSC regulation for cell proliferation and differentiation. The objective of this study was to investigate how significantly gravitational dispersion affects the spontaneously induced osteogenic differentiation of MSCs. Expression of surface antigen was measured by flow cytometry prior to two axial rotational cultures. About 12,500 hMSC cells were spread on culture wells of 1.8 cm(2) surface area and incubated for 7 days at 5% CO(2). The culture medium, 10% FCS/DMEM containing 3 ng/ml bFGF, was replaced every 3 days. Four wells then were placed in a 50-ml centrifugal tube filled with 10% FCS/DMEM without bFGF. The centrifugal tube was attached to the center of the rotor, and two axial rotational cultures were started at 10 rpm each of both rotational speeds. It was confirmed that the hMSCs used in this study expressed typical surface antigens as well as a multipotent differentiation ability for either osteogenic or adipogenic differentiation. Spontaneous expression of alkaline phosphatase (Alp) mRNA following the conventional static culture (1G condition) was suppressed by two axial rotational cultures for 7 days (p < 0.05). A separate study indicated that the cell count number eventually increased from 24,700 ± 6,400 to 78,400 ± 18,700 (p < 0.05). In addition, suppressed Alp mRNA was recovered after an additional 7-day culture under static conditions. This result indicated that dispersion of gravity is a promising modality to regulate osteogenic differentiation of hMSCs.

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.

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.

[Performance evaluation of the CellaVision DM96 system in WBC differentials].

Though differential counting of peripheral blood cells is an important diagnostic tool, this technique requires highly trained staff. Automation of differentials is desirable for economic and time-saving reasons. Recently, the CellaVision DM96 (DM96, CellaVision AB, Lund, Sweden) has been introduced as an automated cell analysis system capable of morphological classification of WBCs and RBCs in pheripheral blood smears. In this study, we routinely analyzed the blood samples from 216 patients by the DM96. The overall preclassification of WBC analysis accuracy value for the DM96 was 90.3%. Good correlation coefficients between final results of the DM96 and manual differentiation were observed. The DM96 system performed high sensitivity and specificity for blasts and immature granulocytes. Although the DM96 system operates more effectively in the normal blood samples than pathological ones, its ability of review slides on the computer screen with a cell-by-cell basis provides real-time collaboration between colleagues when they face the abnormal cells.

[Evaluation of capability of cell count and detection of tumor cells in cerebrospinal and body fluids by automated hematology analyzer].

Sysmex XE-5000 offers the body fluid modus which provides the opportunity to count and differentiate leukocytes in body fluids and cerebrospinal fluid (CFS). In this study, we evaluated the basic performance of this application using routinely obtained samples in comparison with manual counting. Reproducibility study yielded good results in samples with a high white blood cell (WBC) count, whereas relatively high imprecision was observed at low WBC counts. Linearity was established up to 1,500 cells/microL in CFS and 5,600 cells/microL in body fluid. The cell count by XE-5000 was highly correlated with that of the microscopic reference method. Highly fluorescent body fluid cells percent (HF-BF%) was observed in samples with tumor cells or activated macrophages, which provides information about the possible presence of tumor cells. In conclusion, total and differential WBC counts in body fluid and CFS can be reliably determined by XE-5000 in samples with increased cell counts. XE-5000 also provides screening information about the presence of tumor cells for further manual examination.