[Discussion of Routine Laboratory Data of a Patient Complaining of Back Pain and General Fatigue].

Conventional reversed clinicopathological conference (RCPC) is an educational method to interpret labora- tory data. In this RCPC, physicians and several specialists in laboratory medicine discussed laboratory data of a patient with tuberculous spondylitis who complained of back pain and general fatigue. Then, they and the moderators held a question-and-answer session with an audience in a hall, and they tried to understand the detailed state of the patient. This discussion revealed the usefulness of RCPC to elucidate the clinical state of patient. At the same time, we can understand the limits of laboratory data analysis. [Review].

Mean postprandial triglyceride concentration is an independent risk factor for carotid atherosclerosis in patients with type 2 diabetes.

BACKGROUND: Postprandial hypertriglyceridemia is a risk factor for atherosclerotic disease. However, the postprandial triglyceride (PTG) concentration fluctuates markedly and is poorly reproducible. The aim of this study was to determine whether the mean PTG (mean-PTG) concentration is a risk factor for carotid atherosclerosis in patients with type 2 diabetes. METHODS: We measured the fasting and postprandial lipid concentrations, and the maximum intima-media thickness (max IMT) of carotid arteries by ultrasound in 115 diabetic patients. A carotid plaque was defined as max IMT of >1.0mm. The mean-PTG concentration was calculated from several PTG concentrations measured on different days during a 1-year follow-up period. RESULTS: PTG concentrations showed marked intra-individual variability, and ranged from 0.29 to 6.03 mmol/l. Patients with carotid plaques had higher mean-PTG concentrations than those without carotid plaques (1.51 ± 0.57 vs. 1.29 ± 0.47 mmol/l, p=0.025). Neither fasting triglycerides nor one-point PTG concentrations differed between the two groups. Multivariate stepwise logistic regression analysis revealed that the mean-PTG concentration was significantly associated with carotid plaques [OR 1.20 (95% CI, 1.05-1.37), p=0.009], even after adjusting for traditional risk factors including HDL-cholesterol, LDL-cholesterol, age, hypertension, and duration of diabetes. CONCLUSIONS: The mean-PTG concentration is an independent risk factor for carotid atherosclerosis in patients with type 2 diabetes.

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.

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.

Identification of Bcl-2/IgH fusion sequences using real-time PCR and chip-based microcapillary electrophoresis.

BACKGROUND: The determination of polymerase chain reaction (PCR) amplification product sizes of the Bcl-2/IgH fusion gene from follicular lymphoma (FL) provides evidence of clonal identity. METHODS: The present study describes detection of Bcl-2/IgH fusion gene clonality utilizing a small, simple microcapillary electrophoretic chip combined with a real-time PCR method. RESULTS: The microcapillary electrophoretic chip system effectively detects size differences among the Bcl-2/IgH fusion gene amplification products of FL from patient samples; something that is not possible using traditional gel electrophoresis. We also describe the potential of this system to utilize formalin-fixed, paraffin-embedded tissue samples sectioned on charged slides. CONCLUSIONS: The simple detection of Bcl-2/IgH fusion gene clonality using a microcapillary electrophoretic chip provides reliable information for monitoring minimal residual disease of FL, and can be an effective tool for use in clinical laboratories.

[In vitro activity of methylrosaniline chloride (gentian violet) as disinfectant against Candida spp. and Trichosporon spp. isolated from blood samples].

OBJECTIVE: Methylrosaniline Chloride (MRC) is recognized as a disinfectant, but recently is rarely used in the clinic, because of its cytotoxicity when used continuously with conventional concentrations (1% MRC). We have reported the antibacterial activity of MRC with lower concentration against Methicillin-resistant Staphylococcus aureus (MRSA). In this study, we evaluated the antifungal activity of MRC with lower concentrations. MATERIAL AND METHODS: Antifungal activities of MRC against Candida spp. and Trichosporon spp. were tested. All strains tested were isolated from 106 blood or intravenous catheter samples at Juntendo University Hospital from 1995 to 2004. Minimum inhibitory concentrations against fungi were assayed by agar dilution, under both aerobic and anaerobic conditions. RESULTS: A 0.01% or less concentration of MRC solutions showed marked antifungal activity against Candida spp. and Trichosporon spp. under aerobic or anaerobic conditions. CONCLUSION: A 0.01% or less concentration of MRC should be reevaluated for the control of fungal infection and MRSA infection control.

[Bacterial interaction and indirect pathogenesis of Pseudomonas aeruginosa at the growth of MRSA].

Bacterial interactions such as methicillin-resistant Staphylococcus aureus (MRSA) growth inhibition or inactivation of anti-MRSA antibiotics by Pseudomonas aeruginosa as an indirect pathogen were tested by in vitro assay. Paired strains, P. aeruginosa and MRSA, used in this experiment were isolated from 63 respiratory samples at Juntendo University Hospital from 2002 to 2003. Growth inhibitory activities against MRSA by P. aeruginosa were tested with reversed agar plate method. Inactivation of anti-MRSA antibiotics by P. aeruginosa were assayed with disk diffusion method using agar over lay technique. Fifty-six (88.9%) out of 63 samples showed the significant MRSA growth inhibitory activity by co-existed P. aeruginosa. Anti-MRSA antibiotics such as trimetoprim-sulfamethoxazole combination (ST), arbekacin (ABK) and minocycline (MINO) except Vancomycin (VCM) and Teicoplanin (TEIC) were inactivated by the co-existed P. aeruginosa. Our data suggests that P. aeruginosa may play not only as a chronic respiratory pathogen but also as an indirect pathogen. Further, the most P. aeruginosa with anti-MRSA activity isolated respiratory sample may play as a modulator of MRSA infection.

[In vitro indirect pathogenesis of Pseudomonas aeruginosa against anti MRSA chemotherapy].

In the patient with a chronic respiratory disease, both Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) are frequently detected from expectoration. Vancomycin (VCM) and arbekacin (ABK) are both recommended for the chemotherapy of MRSA infection in Japan. Minocycline (MINO) is also selected for the treatment of MRSA infection. While rifampicin (RFP) and a trimetoprim-sulfamethoxazole combination (ST) are also recommended in Europe and USA but not recommended in Japan for the chemotherapy of MRSA infection. It is pointed out that coexistence bacteria affect chemotherapy as an indirect pathogen. Not only an antibacterial action but the immunological action or the metabolic effect against chronic P. aeruginosa infection such as DPB is known by the administration of 14-membered ring macrolides including erythromycin (EM). We considered the influence of P. aeruginosa isolated with MRSA on the activity against anti-MRSA agents by the disk diffusion method with bilayer flat agar in vitro. Moreover, we also examined the influence of EM against the activity of the anti-MRSA agents when P. aeruginosa was coexistence. One strain of MRSA as an indicator strain and 100 strains of P. aeruginosa as test strains, which were obtained from clinical materials, were used for the following experiment. P. aeruginosa was streaked on to the Mueller-Hinton agar culture medium (MHA), and they incubated at 35 degrees C for 24 hours. Then, the blood agar plate was piled up, MRSA was streaked on the blood agar surface, the susceptibility test disks (VCM, ABK, MINO, RFP, ST) were put on it, and incubated at 35 degrees C for a further 24 hours. The diameter of the zone of inhibition around the susceptibility disks against MRSA was measured and compared with P. aeruginosa free experiments. The anti-MRSA activity of MINO, ST and ABK was reduced by coexistence of P. aeruginosa. In RFP and VCM, the anti-MRSA activity was reinforced by coexistence of P. aeruginosa. Although the anti-MRSA activity of ST and ABK has improved by EM addition in the MHA plates, the anti-MRSA activity has not improved in MINO. These results are suggesting that in a MRSA infection, the chemotherapy by anti-MRSA agents were affected by coexistence of P. aeruginosa as an indirect pathogen. The macrolides such as EM may be useful as a modulator for chemotherapy by ST or ABK when MRSA and P. aeruginosa are isolated at the same time from the patient.

[Cell surface and intracellular marker analysis as a laboratory test for the diagnosis of hematological disorders].

Immunophenotyping of hematopoietic malignancies is representative application of cell (surface) marker analysis by flow cytometry and monoclonal antibodies in the clinical laboratory. The multitude of available monoclonal antibodies demands a standardization of the selection and combination of antibodies. Therefore, some international committee or working group proposed the panels or guidelines for the selection of antibodies. Intracellular antigens are of major importance for immunophenotyping of hematological malignancies, and flow cytometric detection of intracellular antigens was improved by the development of new permeabilization/fixation solutions. Recently new gating method was recommended for better isolating the target cells in the flow cytometric analysis. Finally CD55 and CD59 assay for the diagnosis of PNH was mentioned.

Increased circulating CD16+ CD14dim monocytes in a patient with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveoli with a periodic acid-Schiff-positive proteinaceous material. Although the pathogenesis of primary or idiopathic PAP remains unknown, it has been proposed that a deficiency or loss of responsiveness of the monocyte/macrophage lineage to granulocyte-macrophage colony stimulating factor (GM-CSF) is involved in PAP. Secondary PAP is associated with haematological malignancies, especially in myeloid disorders. Herein, we report on an adult with PAP associated with myelodysplastic syndrome (MDS). The CD16+ CD14dim monocytes comprise 5-10% of circulating monocytes in healthy volunteers. Flow cytometric analysis of the patient in the present study revealed increased CD16+ CD14dim monocytes in the peripheral blood. It has been demonstrated that the expression of CD16 and CD14 is regulated by macrophage colony stimulating factor (M-CSF) and GM-CSF. Hence, serum cytokines were analysed in our patient and the concentration of serum GM-CSF was found to be less than the lower limit of the assay. In addition, serum M-CSF and granulocyte colony stimulating factor levels were only slightly increased above the normal range. These results suggest that the increase in the CD16+ CD14dim subpopulation in the circulation of our patient indicates another pathogenetic mechanism for secondary PAP, such as hyperresponsiveness of the monocyte/macrophage lineage to these cytokines.

Evaluation of hematological values obtained with reference automated hematology analyzers of six manufacturers.

We evaluated assays of the same fresh blood samples with six different types of reference automated hematology analyzers developed by the following manufacturers: Beckman Coulter, Sysmex, Bayer, Abbott, Nihon Kohden and Horiba. Fresh whole blood samples treated with dipotassium ethylenediaminetetraacetic acid (EDTA K2) were collected from three healthy adult volunteers. The complete blood counts (CBC) including red blood cell count (RBC), hemoglobin (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), white blood cell count (WBC), platelet count (Plt), reticulocyte percentage (Ret) and leukocyte differential counts including % neutrophils (Neu), % lymphocytes (Lym) and % monocytes (Mon) were surveyed with a reference automated hematology analyzer from each manufacturer. The process from sampling to analysis was performed according to procedures in hospital clinical laboratories. RBC, Hgb, Hct and MCV exhibited allowable differences within 5% of mean value among all instruments. Large differences greater than 10% of mean value in WBC, Neu and Lym between Horiba and other manufacturers, and in Plt between Nihon Kohden and other manufacturers, were observed. Ret and Mon exhibited large differences over 10% of mean value among almost all of the instruments tested. This survey suggests that all parameters exhibiting differences greater than 10% of mean value among instruments should be improved for clinical use to ensure good external quality control in blood cell counting and leukocyte differential counting using automated instruments.