Single nucleotide polymorphisms of the DGKB and VCAM1 genes are associated with granulocyte colony stimulating factor-mediated peripheral blood stem cell mobilization.

We previously reported the association between LDL cholesterol level (LDL-C) and granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (PB) hematopoietic stem cells (HSC). In this study, we investigated the association between gene single nucleotide polymorphisms (SNPs) involved in hematopoiesis and lipid level and PBHSC mobilization. In 46 patients who underwent peripheral blood stem cell harvest (PBSCH), we measured CD34-positive cells in PB and PBSCH, and the patients were classified into good, intermediate, or poor mobilizer groups based on the CD34-positive cell counts. And SNPs of the OR4C12, ENO1, RERE, DGKB, DSC3, VCAM1, CD44, and FADS1 genes were investigated. The frequency of the TT type of the DGKB gene was higher in the poor mobilizer group compared to other groups (p<0.05), whereas that of the CC type of the VCAM1 gene was high in the good mobilizer group (p<0.05). Association with the efficiency of HSC mobilization to PB were found in the SNPs of the DGKB gene involved in cell transport and SDF-1-induced migration ability and of the VCAM1 gene which is essential for HSC homing, suggesting that SNPs involved in cell migration ability might be partly involved in HSC mobilization to PB.

[Usefulness of the Loopamp Mycoplasma P Detecting Reagent Kit developed based on the LAMP method].

Pathogenic bacteria of Mycoplasma pneumonia, Mycoplasma pneumoniae, do not respond to ß-lactam antimicrobial agents. Therefore, it is clinically important to promptly identify infection with this type of bacteria. However, conventional examination methods such as culture and antibody tests are not useful for the rapid diagnosis of this bacterial type. In this study, we examined a Loopamp Mycoplasma P-detecting Reagent Kit developed based on the LAMP (Loop-mediated isothermal amplification) method, which may overcome the limitations of the conventional methods. The consistency rate for the conventional polymerase chain reaction (PCR) method was 98.3%. That for a rapid antibody test, immunocard Mycoplasma antibody, was 54.0%, suggesting the limitation of the rapid antibody test. We compared a pretreatment method using a Loopamp PURE DNA Extraction Kit, as a simple DNA extraction method, with that using a QIAamp DNA Mini Kit. The consistency rate was 91.4%, suggesting the usefulness of the simple DNA extraction method. The use of this kit may facilitate a more rapid diagnosis. The Loopamp Mycoplasma P-detecting Reagent Kit is useful for the accurate and rapid diagnosis of Mycoplasma pneumoniae infection.

[Usefulness of phage ORF typing, a rapid genotyping method as a molecular and epidemiological method for detecting methicillin resistant Staphylococcus aureus].

Surveillance is very important for preventing the nosocomial spread of methicillin-resistant Staphylococcus aureus (MRSA), and the pulsed-field gel electrophoresis (PFGE) method has long been used to identify the infection source and route as a molecular and epidemiological genotyping method. However, the use of the method in routine clinical laboratory measurements is difficult due to its complicated procedures. Since a molecular and epidemiological genotyping kit based on the POT (Phage Open-reading Frames Typing) method has been developed, we examined 192 MRSA isolates newly detected from inpatients in our hospital in 2010 in order to investigate the usefulness of POT for surveying outbreaks of MRSA. Among the 192 isolates 118 were suspected of nosocomial spread by the previous method, which defined a MRSA detection at more than 48 hours after admission as a nosocomial spread. The POT method was introduced at our laboratory in 2010, and we were able to recognize 38 patients as having strongly suspected nosocomial MRSA infection with the POT method taking into consideration the infection situation, such as places (wards and transfer) and time (date of admission and date of collected samples). Our Infection Control Division was confidently able to demonstrate the current condition of the nosocomial spread by providing the results to the clinical staff, who were also able to practice infection control confidently. We concluded that the POT method was very useful and convenient for investigating MRSA isolates and evaluating collected data because no particular analysis other than the digitizing electrophoretic pattern method was necessary.

[Relativity in multiple myeloma and KL-6].

KL-6 is a high-molecular-weight mucinous glycoprotein discovered as a pulmonary adenocaricinoma related antigen. Its levels are used as a biomarker of lung injury in interstitial pneumonia. We here report a case of multiple myeloma with Bence-Jones lambda type whose serum KL-6 level was revealed high at a concentration of 19,400 U/ml. Next, we analyzed the blood test profiles and the concentrations of KL-6 in 20 patients with multiple myeloma. CD19/CD56 double negative fraction on myeloma cells with high expression of CD38 was found in all 5 patients with multiple myeloma having elevated KL-6 level. Patients with interstitial pneumonia show high level of KL-6. We, therefore, need to differentiate the interstitial pneumonia and the above-mentioned multiple myeloma when serum KL-6 level is high.

[One example of false negative hepatitis B surface antigen (EIA) result due to variant S area strain and reagment reactiveness related to hepatitis B surface antigen].

The presence in serum of the Hepatitis B surface antigen (HBsAg), the outer envelope of the hepatitis B virus (HBV), indicates viral infection, used in laboratory tests to confirm this. We report a case of discrepancy among HBsAg test results detected between measurements in a subject with HB infection. Gene analysis demonstrated several S region gene mutations, not detected previously. We tested 12 measurements e.g., EIA, CLIA, CLEIA, F-EIA, MAT, and IC for whether they could detect our subject's HBsAg and found that it was not recognized by a method using only a single monoclonal antibody to detect HBsAg in two detection processes, in contrast to the 11 other measurements, which used two different antibodies. This case shows that amino acid substitution may cause a false negative result for HBsAg. Gene mutations known to occur in HBV, should thus trigger an awareness of the need to keep in mind that false negative results can happen in case such as ours.

[Approach for the improvement of microscopic urinary sediment examination among the hospitals in using multifocal virtual software].

Microscopic examination is usually used for analysis of urinary sediment. The problem of it is difference of the examination results among hospital's clinical laboratories. Photo-survey, training conference using photographs and seminars are held for each examiner to obtain accurate results. However, diagnosis of urinary sediment with photographs is difficult because detailed 3-dimensional cell morphology of urinary sediment cannot be analyzed only with photographs. It is also difficult to have opportunity for all examiners to get enough times of real microscopic training. To evaluate the availability of multi-focal virtual software, diagnosis of urinary sediment under 17 hospital's laboratories was compared between multifocal virtual software survey and conventional photo-survey. Questionnaires were also filled out by the examiners. The rate of correct answers to virtual software survey was 73.9% that was almost same as the result of the survey by Saitama Sightron Society. The rate of correct answers by virtual software survey was higher than photo survey. As a result of questionnaires, the participants take certain advantages of virtual software in spite of several problems. In conclusion, multifocal virtual software enables an accurate diagnosis of urinary sediment in 3-dimension, which leads to reduce the discrepancies of examination results among laboratories.

Effective ex vivo expansion of hematopoietic stem cells using osteoblast-differentiated mesenchymal stem cells is CXCL12 dependent.

Effective ex vivo expansion of hematopoietic stem cells (HSCs) is a prerequisite for HSC transplantation. Growth and maintenance of HSC is dependent on cytokine and niche factors. We investigated whether mesenchymal stem cells (MSCs) or osteogenic cytokine-differentiated MSCs play a role in HSC expansion. We used the human HM3.B10 (B10) MSC cell line and the osteoblast-differentiated B10 (Ost-B10) as a feeder layer and examined ex vivo expansion of CD34(+)CD38(-) HSCs obtained from peripheral blood (PB) and cord blood (CB) with or without several growth cytokines. Both undifferentiated B10 and Ost-B10 cells exhibited similar effects on total HSC expansion; however, Ost-B10 demonstrated a higher potency in CD34(+)CD38(-) cell-specific proliferation in the presence of cytokines compared to undifferentiated B10 HSCs. Colony-forming cell assay and long-term culture initiating cell assay revealed that Ost-B10 displayed multipotent differentiation and enabled long-term ex vivo culture of HSCs. We next examined the relationship between HSC expansion and the presence of various chemokines. CXCL4 and CXCL12 expression were increased in Ost-B10 cells compared with the B10 cells. CD34(+)CD38(-) cells were significantly increased with CXCL12, but not CXCL4 treatment. siRNA inhibition of CXCL12 decreased CXCL12 secretion in both B10 and Ost-B10, whereas expansion of CD34(+)CD38(-) cells was decreased in Ost-B10 alone. These results demonstrated that ex vivo expansion of HSCs may be highly effective through osteoblast-differentiated MSCs acting as a feeder layer, and likely operates through the CXCL12 chemokines signaling pathway.

Quantification of CSF cystatin C using liquid chromatography tandem mass spectrometry.

BACKGROUND: Cystatin C (CST3), a ubiquitously expressed cysteine protease inhibitor, is implicated in several neurological diseases. Here, we have developed an accurate CST3 measurement system based on liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: LC-MS/MS based measurement for CSF CST3 was validated by determination of assay precision, accuracy and recovery. The values were compared with those measured by immunoassay. Glycosylation of CST3 in CSF was analyzed by Western blotting and lectin blotting. RESULTS: Measuring standard CST3 by LC-MS/MS produced a linear standard curve that correlated with assigned values (r2=0.99). Both intra- and inter-assay variation was <10%. Although showed a correlation, the average CST3 concentration measured by LC-MS/MS was significantly higher than that of immunoassay. Western blotting showed the presence of a 25KDa species along with CST3 monomer (14KDa) in CSF. The volume of 25KDa species was decreased by deglycosylation. Lectin blotting revealed a 25KDa glycosylated protein in sialidase-treated CSF, which was decreased by deglycosylation. However, deglycosylation did not alter CST3 concentration measured by immunoassay. CONCLUSIONS: Our results suggest that LC-MS/MS-based CST3 measurement is a robust method with higher detection ability. Such method could be useful for the diagnosis and monitoring of neurological diseases.

Effects of six functional SNPs on the urinary 8-isoprostane level in a general Japanese population; Shimane COHRE Study.

Oxidative stress is an important risk factor for cardiovascular diseases. Although a variety of genetic factors are assumed to contribute to the regulation of oxidative stress, evidence in human populations is insufficient. In this study, we therefore evaluated the effects of six functional single-nucleotide polymorphisms (SNPs) on the oxidative stress under a cross-sectional study design. Participants of the health examination in two neighboring counties were recruited in a mountainous region of Shimane prefeture, Japan (n=1092). As a marker for the oxidative stress, the urinary 8-isoprostane (IsoP) was measured by ELISA. The six SNPs were genotyped using the Taqman method. None of the SNPs showed a significant effect on the IsoP level. However, the Generalized Multiple Dimensionality Reduction (GMDR) method identified that the combination of the two SNPs, MTHFR C677T and eNOS T-786C, showed a significant effect on the IsoP level in this population. The linear regression analysis confirmed that the high risk genotype identified in the GMDR was an independent factor influencing the IsoP even after adjustment of confounding factors. This result suggested that GMDR analysis might be useful to identify concealed effects of combined SNPs.