Heavy Element Effects in the Diagonal Born-Oppenheimer Correction within a Relativistic Spin-Free Hamiltonian.

Methodologies beyond the Born-Oppenheimer (BO) approximation are nowadays important to explain high precision spectroscopic measurements. Most previous evaluations of the BO correction are, however, focused on light-element molecules and based on a nonrelativistic Hamiltonian, so no information about the BO approximation (BOA) breakdown in heavy-element molecules is available. The present work is the first to investigate the BOA breakdown for the entire periodic table, by considering scalar relativistic effects in the Diagonal BO correction (DBOC). In closed shell atoms, the relativistic EDBOC scales as Z(1.25) and the nonrelativistic EDBOC scales as Z(1.17), where Z is the atomic number. Hence, we found that EDBOC becomes larger in heavy element atoms and molecules, and the relativistic EDBOC increases faster than nonrelativistic EDBOC. We have further investigated the DBOC effects on properties such as potential energy curves, spectroscopic parameters, and various energetic properties. The DBOC effects for these properties are mostly affected by the lightest atom in the molecule. Hence, in X2 or XAt molecule (X = H, Li, Na, K, Rb, and Cs) the effect of DBOC systematically decreases when X becomes heavier but in HX molecules, the effect of DBOC seems relatively similar among all the molecules.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2009].

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 2655 strains including 810 strains of Gram-positive bacteria, 1635 strains of Gram-negative bacteria, and 210 strains of anaerobic bacteria obtained from 30 medical institutions during 2009 was examined. The results were as follows; (1) MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multidrug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). (2) MEPM maintained potent and stable antibacterial activity against Pseudomonas aeruginosa. The proportion of MEPM-resistant strains to ciprofloxacin-resistant strains or imipenem-resistant strains were 53.1% and 58.0% respectively. (3) The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (26 strains) in enterobacteriaceae. And the proportion of metallo-beta-lactamase strains was 2.0% (6 strains) in P. aeruginosa. (4) Of all species tested, there were no species except for Bacteroides fragilis group, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 14 years passed after available for commercial use in Japan.

[Evaluation of serum iron in hemodialysis patients and its variation due to the difference of blood sampling time].

Diurnal variations in serum iron concentration were examined to investigate the influence of sampling time in hemodialysis (HD) patients and healthy subjects. The serum iron concentration and TIBC of HD patients decreased significantly (p<0.01, p<0.01 respectively) compared to those of healthy subjects. Inversely, the serum ferritin concentration of HD patients increased significantly (p<0.01) compared to that of healthy subjects. These findings show the disturbance of iron transport system: under such condition intracellural iron transition out into peripheral blood stream is low in HD patients. Serum iron concentration in samples collected in the evening decreased significantly both in HD patients (p<0.05) and in healthy subjects (p<0.01). Diurnal variations in serum iron concentration reveal almost similar decrement in both groups. In HD patients, serum iron concentration of the blood samples collected on the third day morning after HD and second day morning after HD was examined to see the influence from changes of circulating plasma volume. The serum iron concentration and Hct value in the second day sampling increased significantly compared to the third day sampling (p<0.01, p<0.01 respectively). In addition, the serum iron concentration corrected by Hct in the second day sampling increased significantly (p<0.01) compared to the third day sampling. We conclude from our results that diurnal variations of serum iron concentration vary in sampling time in HD patients as well as in healthy subjects. We also deduct that there may be other factors concerning changes in circulating plasma volume.

[Utility of Hybrisep in the diagnosis of blood stream infection].

Hybrisep is an in situ hybridization (ISH) method to detect phagocytosed bacteria in peripheral blood neutrophils and macrophages. We report 10 actual clinical cases tested using Hybrisep with new DNA probes, and the data were compared to the actual blood culture results. A normal Hybrisep strategy employs 5 DNA probes to detect the following bacterial DNA: "SA" probe for S. aureus, "SE" for S. epidermidis, "PA" for P. aeruginosa, "EF" for E. faecalis, and "EK" for E. coli, E. cloacae, and K. pneumoniae. Six newly designed DNA probes were used in this study: "GB" probe for 49 common bacteria, "SP" for S. pneumoniae, "BF" for B. fragilis, "HI" for H. influenzae, "GC" for Candida species, and "CA" for C. albicans. Three cases were positive on ISH, but all their blood cultures were negative. One case showed a positive blood culture, but was negative on ISH. In another 6 cases, both were negative. We postulated that empirical therapy of antibiotics resulted in only positive ISH. Cases only showing a positive outcome on blood culture might be due to a diminished phagocytic function during patients' severe disease conditions. In conclusion, ISH with Hybrisep has clinical advantages such as being able to defect causative pathogens even after the use of antibiotics, and facilitates more rapid identification than routinely performed bacterial cultures only.

[Adaptations to the incidental cases encountered at the division of clinical laboratory].

We analyzed 4 cases that were not determined as incidents and another 7 cases determined as incidents, found at the department of clinical laboratory of us from April 2009 to March 2010. The former cases were, excess values of LD, and Cl, and glucose leveled as 0 mg/dl incorrectly, and misdirected blood samples at the ER. Our routine equipment and sample flow did not detect these false values. Resetting for auto dilution system and secondary check by every worker were reconsidered for these measurements. Antiseptic drug usage was notified by clinicians, and actually affected to the excessive Cl value. Real incidents were, two unprocessed samples, leakage of a sample, missing processes that caused delay of clinical practice, mixed up sample labels, a lost narcotic patch during cardiac ultrasonography. A lack of checking, carelessness, and accidental mistakes were reevaluated and reminded for workers on the duties. Also inadequate pharmaceutical knowledge and responsibilities of this section might severely affect on these lessons. Efforts were taken so that all workers shared the accurate information. Code blue cases are defined here as those of life-threatening events and sudden vital changes occurred in highest emergency, involve all health care workers, patients, and families. It is very important here to keep regular trainings for workers to cope with such events as well as preemptive assessments on environment and underlying risks in our laboratory. In line with the continuous advances in clinical medicine, medical safety managements are growing issues. To achieve safer environment and minimize various type of risks in the hospital, these incidents are to be assessed and reported regularly.

[Various troubles in the Central Clinical Laboratory (violence, others) and actions to be taken].

We need to attend to a patient face to face in the physiological function testing room and the central blood-drawing room of the central clinical laboratory in a hospital. Recently, there have been some problems and troubles experienced by medical staff because patients may have had some over-expectations regarding our medical service and their sense of entitlement may have been unrealistic. We report some problems experienced in our Central Clinical Laboratory and hospital, and describe some solutions. If a medical staff member gets into trouble with a patient because of some difference in awareness between him/her and the patient, we should take prompt action regarding these claims and be considerate to the patient. However, we have to take a tough stance against some severe cases, for example violence and intemperate language directed toward medical staff. One of the most important things is to investigate complaints against us, and, through this, keep on improving. Another important thing is to disseminate information among central clinical laboratory teams liaising with nursing and other departments. Therefore, we can establish a good trusting relationship between patients and staff, and consequently our patient service will improve.

[Nationwide survey for autoantibodies and proposal of a new method for standardization of rheumatoid factors].

There exist inter-laboratory differences in measurements of rheumatoid factor (RF) and antinuclear antibodies (ANA), leading to a misdiagnosis of rheumatoid arthritis (RA) and other collagen diseases. This study was carried out to bring the positivity of RF and ANA of different reagents into accord by standardizing their data. The titer and cutoff value was inconsistent among the 17 different kits. We found a possibility in standardization of RF by a new concept in cooperation with Japanese Committee for Clinical Laboratory Standards (JCCLS). Sera from 1300 healthy subjects and 79 RA patients were measured for RF by 17 different RF kits, and sera with a little deviation among the kits were selected. Panels for detection of RF positive rate in healthy subjects were made from the pooled sera. The cutoff value in 5% positive in the panel was defined tentatively as 15 IU/ml. The 100 IU/ml was also able to become in general accord by adjusting the individual data using pooled RF-positive reference sera. The nationwide survey of immunofluorescence ANA (IF-ANA) was performed in 41 laboratories using 6 pooled sera. The reported titer was fairly different among laboratories, and a striking discrepancy was found for low-titer samples. When the titer was corrected by simultaneously measured reference serum, inconsistency of ANA titer among different laboratories was mostly compensated. Here, we propose a new method for standardization of RF and also try to standardize the positivity of RF and IF-ANA by providing pooled reference sera.

[Induction of PBP2' by antibiotics and disinfectants in MRSE].

OBJECTIVE: Methicilllin-resitant Staphylococcus aureus (MRSA) is still the most important bacterium for hospital infection control, and is known to exhibit beta-lactam resistance. Moreover, the increase in PBP2'-producing methicillin-resistant coagulase-negaive Staphylococcus (MR-CNS), especially methicillin-resistant S. epidermidis (MRSE) has been problematic. In this study, we investigated the induction of PBP2' by MPIPC, other antibiotics and disinfectants in MRSE. MATERIALS AND METHODS: The bacterial strains used were MRSE isolated in our clinical laboratory. MRSA-LA 'Seiken' was used for the detection of PBP2'. To investigate induction of PBP2' by MPIPC in MRSE, MRSE was cultured on the medium containing MPIPC at 11 different concentrations from 0.0001 to 6 microg/ml, and PBP2' induction was investigated. Strains in which no induction was noted at a low MPIPC concentration were cultured with other antibiotic discs and discs impregnated with various disinfectants, and PBP2' was detected in colonies that grew around the disc and PBP2' induction was investigated. RESULTS: In the culture on MPIPC-supplemented medium, PBP2' was detected in all strains at 0.01-6 microg/ml. At 0.001 and 0.0001 microg/ml, 8/10 and 4/10 were positive, respectively. Addition of another beta-lactam, particularly cephem antibiotics, induced PBP2' in some strains that were negative at 0.0001 microg/ml. In cultures with disinfectants, inhibition zones were noted, but no PBP2' was induced. CONCLUSION: PBP2' was induced by a low beta-lactam and was not by disinfectants in MRSE.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2006].

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 876 strains of Gram-positive bacteria, 1764 strains of Gram-negative bacteria, and 198 strains of anaerobic bacteria obtained from 30 medical institutions during 2006 was measured. The results were as follows; 1. MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus. 2. As for Pseudomonas aeruginosa, all of the MEPM-resistant strains were resistant to imipenem (IPM). MEPM showed low cross-resistant rate both againt IPM-resistant P. aeruginosa (41.8%) and ciprofloxacin-resistant P. aeruginosa (33.3%). 3. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 4.3% (6 strains) in Escherichia coli, 1.1% (1 strain) in Citrobacter freundii, 21.7% (5 strains) in Citrobacter koseri, 3.1% (4 strains) in Klebsiella pneumoniae, 3.3% (3 strains) in Enterobacter cloacae, 0.8% (1 strain) in Serratia marcescens, and 4.9% (2 strains) in Providencia spp. The proportion of metallo-beta-lactamase strains was 3.1% (10 strains) in P. aeruginosa. 4. Of all species tested, there were no species, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem at present, 11 years after available for commercial use.

[Evaluation of the measurement of waist circumference].

OBJECTIVES: A series of basic analyses was performed to disclose the problems in the measurement of waist circumference (WC) at umbilical level, which will provide quite important parameters in the national annual health check for metabolic syndrome coming in April, 2008 in Japan. MATERIALS AND METHODS: WC and body fat ratio was measured in 53 healthy volunteer, especially in 10 subjects (male 10, female 10), measurements were done by 5 examiners identically to disclose inter-examiner variances, changes due to respiration maximal depression of abdomen (or maximal intentional depression of WC), and eating and after lunch. RESULTS AND DISCUSSION: WC tended to be higher in aged male, than aged and young female, and was positively correlated with body fat ratio. Minimal and maximal inter examiner variances of WC were 0.3 and 4.7cm, respectively. We need to pay attention to it. The effect of respiration and eating lunch were relatively low and may not cause serious problems. Intentional depression of abdomen caused from 2 to 8.1cm decrease of WC, which could provide false negative results. CONCLUSION: WC measurement seems to be easy but actually it is difficult. We need to remind some difficulties or preanalytical or analytical problems in this easy looking examination, especially used in large scale screening test.

[Preanalytical issues].

Preanalytical issues in laboratory medicine have been recognized to be important but not so much effort have been focused to them. Urinalysis is one of the most susceptible examinations to preanalytical issues because urine samples are collected by patients themselves and so is stool examination too. Midstream urine catch is well-known appropriate procedure for many urinalysis. Surface scratching is an appropriate method of stool occult blood testing for colorectal cancer and also strategies to avoid the contact of stool with toilet water is another important technique. We also report a case of abnormal results of creatinine clearance (Ccr) caused by the urine collection apparatus as an example of preanalytical issue.

[Sampling variables in examinees--skin site differences in the measurements of bleeding time and self-monitoring of blood glucose].

Differences in bleeding time and self-monitoring blood glucose (SMBG) level at various skin sites have been scarcely examined in humans. The within-run variation (n=6) of bleeding time in earlobes (Duke's method) ranged from 1 min to 3 min and 30 sec (mean = 1 min and 40 sec), and in day-to-day variation (n= 8), it ranged from 1 min to 3 min (mean = 1 min and 36 sec). Site difference in bleeding time was speculated. In SMBG, firstly, sequential measurement of blood collected from 10 sites in the left forearm was performed, and secondly, comparative measurements of venous blood and blood from finger tips (1-5th), palm (3 sites), forearm (4 sites) and earlobe (1 site) were sequentially performed within 30 min before and after glucose (Trelan-G, 75g) intake. Glucose levels in blood from the fingertips, palm and forearm were generally higher than that of venous blood, and site differences were observed among fingertips, palm and forearm. It was speculated that bleeding time and capillary blood glucose or SMBG level differ among skin sites.

[Standardization and issues of rheumatoid factor measurement].

OBJECTIVES: To disclose the current situation of rheumatoid factor (RF) measurement in Japan. METHOD: A small-scale survey was performed among members of the committee for the improvement of RF measurement in 2003 using manufacturers' RF reference materials valued based on WHO RF reference and pooled sera. A nationwide questionnaire survey was also performed. Questionnaires were sent to 356 educational institutes certified by the Japan College of Rheumatology in December 2004. RESULTS: The small-scale survey showed inter-laboratory differences of measured RF values, especially using RF references, and less in pooled sera. In the questionnaire survey (187 responses, recovery, 53%), qualitative or semi-quantitative RF measurement methods were used in a small number of institutes and quantitative methods were used in large numbers of institutes, the latex immunoassay (turbidimetric or nepherometric) was predominantly employed. The measuring instruments were various. The upper limit of the reference interval was distributed widely from 5 IU/ml to 40 IU/ml, indicating obvious inter-laboratory differences in RF measurement. Many institutes used the reference intervals recommended by the manufacturers. CONCLUSION: There are remarkable inter-laboratory differences in RF measurement. Clearly, one of the major issues is the lack of reference materials. We therefore need to establish a reference material of RF, even if it is a temporary one. Scientific societies (Japan College of Rheumatology and Japanese Society of Laboratory Medicine) and manufacturers providing RF measurement reagents should work together for the improvement of RF measurement.

Serum amyloid A (SAA) concentration varies among rheumatoid arthritis patients estimated by SAA/CRP ratio.

BACKGROUND: C reactive protein (CRP) and serum amyloid A (SAA) are similarly sensitive indicators of inflammation, but discrepancies are recognized in several disease conditions. SAA levels are generally higher and vary more among individuals. The significance of the SAA/CRP ratio was evaluated in the estimation of SAA. METHODS: Sera of out-patients with rheumatoid arthritis (RA) and healthy subjects were measured for CRP and SAA cross-sectionally and longitudinally by a highly sensitive latex agglutination turbidimetric immunoassay. The results were classified into four groups according to the CRP concentration, and the SAA/CRP ratios were calculated and evaluated. RESULTS: In the cross-sectional study, CRP and SAA were shown to correlate significantly in sera of patients with RA, but not in healthy subjects. The SAA/CRP ratios were wide RA patients and tended to decrease in the range and mean values were observed depending on the concentration of CRP. In the longitudinal study, variations of the ratio among individuals were wide, but smaller and similar within individuals. The difference in magnitude of the ratios is due to the difference in SAA concentration. CONCLUSION: Variations of SAA/CRP ratios were affected by SAA concentrations. The ratio should be checked in the evaluation of SAA.

Proteomics approaches to identify tumor antigen directed autoantibodies as cancer biomarkers.

The identification of autoantibodies to tumor cell proteins by proteomics approaches has great potential impact on cancer biomarker discovery. The humoral immune response represents a form of biological amplification of signals that are otherwise weak due to very low concentrations of antigen, especially in the early stages of cancers. In addition, proteomics can detect immunoreactivity directed against protein post-translational modifications. Two-dimensional gel based Western blots, protein antigen microarrays, and multiplex ELISA reactions have been applied by our group to antigen based biomarker detection and validation. The latter two are based on liquid-phase separations that are suitable for automation. This work has resulted in the identification of numerous cancer biomarker candidates. Large clinical studies are currently planned to establish their value in early cancer diagnosis.

Reactivity of agalactosyl IgG with rheumatoid factor.

BACKGROUND: The difference in the reactivity of rheumatoid factor (RF) with agalactosyl IgG and galactosyl or intact IgG is controversial. METHODS: ELISAs for IgM-RF, using agalactosyl IgG and galactosyl IgG as a coated antigen, were developed. The sera of patients with RA, systemic lupus erythematosus (SLE), chronic liver diseases (CLD) and healthy subjects were measured for IgM-RF. An absorption test was performed using these IgGs with or without heat-pretreatment to determine any differences in the antigenicity between agalactosyl IgG and intact IgG. RESULTS: IgM-RF titers were higher against agalactosyl IgG than those against both heated-intact and intact IgG in three of nine RA patients' sera. Three other serum samples showed similar titers against all three forms of IgG and the remaining three serum samples showed varied results. IgM-RF titers were higher in sera of RA patients than those of SLE, CLD and healthy subjects. Absorptions of RA sera showed that RF was completely absorbed by 5x of absorptions with agalactosyl IgG as well as intact IgG. CONCLUSIONS: The reactivity of IgM-RF is higher with agalactosyl IgG than with heated-intact or intact IgG. The antigenic epitopes of agalactosyl and intact IgG were shown to be closely similar by the absorption test.

Potassium flux of erythrocytes in chronic hemodialysis patients.

BACKGROUND: In chronic hemodialysis patients, hyperkalemia is frequently observed. In these patients, erythrocytes were examined to know whether they participate in the regulation of K(+) or not. METHODS: Erythrocyte K(+) release (DeltaKr) was induced by the incubation of erythrocyte suspension at 4 degrees C for 24 h and the K(+) influx followed at 37 degrees C for 3 h. K(+) flux of erythrocytes or DeltaKi/DeltaKr ratio, which was reflected by Na(+)/K(+)-exchanging ATPase, was measured in chronic hemodialysis patients. K(+) concentration was measured by ion-selective electrode method. RESULTS: Non-diabetic hemodialysis patients classified into three groups according to their serum levels were compared for various factors. Among them, the DeltaKi/DeltaKr ratios in medium- and high-serum K(+) groups were significantly lower than those in the low serum K(+) group. The effect of hemodialysis on erythrocyte K(+) flux was examined. After hemodialysis, the mean DeltaKi/DeltaKr ratio increased significantly compared with that before the treatment. Erythrocyte K(+) concentrations converted into a narrower range after hemodialysis. CONCLUSION: The reduced K(+) flux in erythrocyte may play a part in the development of hyperkalemia in non-diabetic chronic hemodialysis patients.

Identification of a dysfibrinogenemia of gammaR275C (Fibrinogen Fukushima).

BACKGROUND: Various dysfibrinogenemias have been identified worldwide. This paper describes a case of dysfibrinogenemia recently identified in our laboratory. PATIENT: A 34-year-old pregnant woman without any clinical complaints was admitted to our hospital for delivery. She had an extremely low fibrinogen concentration as determined by the thrombin time method though immunoassay showed a titer within the reference range. Dysfibrinogenemia was suspected and further analyses were performed including on her family. Thrombin time was measured using human and bovine thrombin with and without calcium ion. Reptilase time was also measured. To identify the genetic mutation responsible for this dysfibrinogen, genomic DNA extracted from the blood was analyzed for mutation-rich regions in the fibrinogen gene. RESULTS: The subject, her mother and her two infants showed the same pattern of results while her father showed a regular pattern. Thrombin time calculated using both human and bovine thrombin and reptilase time was elongated in the propositus. The extent of the elongation was decreased in the presence of calcium ion. DNA sequencing showed heterogeneous fibrinogen gammaR275C mutations in the propositus, mother and two children. The father showed no mutation. CONCLUSIONS: A case of dysfibrinogenemia gammaR275C without any clinical symptoms was found by routine coagulation testing and was genetically identified.