Air contamination of therapeutic drug monitoring assay reagents results in falsely high plasma ammonia levels.

BACKGROUND: Accumulating evidence shows that contamination of blood samples by atmospheric ammonia affects blood ammonia test levels; however, reports on the effect of ammonia contamination of assay reagents are limited. Here, we aimed to clarify the effect of ammonia contamination of assay reagents, particularly the therapeutic drug monitoring (TDM) reagents, on the detection levels of blood ammonia using enzymatic assays. METHODS: Ammonia gas was measured in the refrigerator compartment of the automatic analyser and the reaction tank water, probe wash water and drain outlets connected to the automatic analyser. At different time points following the closure of the cold storage, ammonia levels in quality control plasma samples were measured using three commercial assay reagents to evaluate the effect of air contamination. The distribution of evaporated ammonia in the reagent was measured using the CicaLiquid NH3 assay kit containing the assay reagent most affected by air contamination. RESULTS: It was confirmed that ammonia gas was generated in the cold storage of the automatic analyser. More than half of the reagents detected >0.25 ppm ammonia, and the highest concentration was detected in the TDM reagent. The ammonia levels obtained using all three reagents increased significantly after 3 h of air contamination. The effect was resolved by measuring a 'dummy' sample or mixing the reagents by inversion. CONCLUSIONS: We demonstrated that air contamination by TDM reagents placed in cold storage could result in significantly falsely high ammonia measurements. Preventing this effect would improve the accuracy of ammonia measurements.

Bone marrow ring sideroblasts in hematological diseases: an analysis of consecutive 1300 samples in a single institution.

The incidence of MDS-RS in Japan has been recognized as about 5% which is lower than that in European countries. Insufficient use of iron staining tests in Japan has been noted as one conceivable factor contributing to this apparently lower prevalence. To investigate this issue, we analyzed the proportion of ring sideroblasts (RS) in 1300 bone marrow samples from patients with hematological diseases at Kitasato University Hospital, including iron staining of all samples. Sixteen of 96 patients with MDS (16.7%) were diagnosed with MDS-RS, and this accounted for 26.2% of MDS without excess blasts. Some MDS-EB (22.9%) and AML-MRC (13.8%) patients also had ≥ 15% RS. In contrast, RS were rarely found in myeloid neoplasms without dysplasia and non-myeloid diseases: only 1 in 46 (2.2%) patients with AML without dysplasia, 2 in 93 (2.2%) with MPN, and 8 in 984 (0.8%) with non-myeloid diseases had ≥ 5% RS. These results indicate that prevalence of MDS-RS in Japan may be higher than conventionally recognized and that RS are principally restricted to myelodysplastic disorders. Further multicenter studies using consecutive bone marrow samples with iron staining tests will be required to confirm our findings.

Portal hemodynamic effects of lenvatinib in patients with advanced hepatocellular carcinoma: A prospective cohort study.

AIM: Lenvatinib is an oral, multitargeted, tyrosine kinase inhibitor, which suppress tumor angiogenesis and tumor progression. It was non-inferior to sorafenib in overall survival in untreated advanced hepatocellular carcinoma (HCC). Sorafenib had a beneficial effect on portocollateral circulation with portal hypertension in translating and clinical studies. However, the hemodynamic effects of lenvatinib appear to be different from those of sorafenib because the efficacy of lenvatinib for vascular endothelial growth factor receptors and fibroblast growth factor receptors is different from that of sorafenib. This study was prospectively performed to evaluate the portal hemodynamic effect of lenvatinib in patients with advanced HCC using duplex Doppler ultrasonography. METHODS: In total, 28 Child-Pugh class A or B patients with advanced HCC received lenvatinib depending on body weight daily for 2 weeks. Primary outcomes were changes in the hemodynamics of the portal venous system using duplex Doppler ultrasonography before and after the 2-week administration of lenvatinib. RESULTS: The portal venous flow velocity (cm/s) significantly reduced (27 ± 12.1 vs. 22.6 ± 8.0, P = 0.019), while portal venous area (cm2 ) did not change after the 2-week administration (0.80 ± 0.36 vs. 0.82 ± 0.27, P = 0.665). Therefore, the congestion index (portal venous area/portal venous flow velocity), which reflects the pathophysiological hemodynamics of the portal venous system significantly worsened (0.037 ± 0.025 vs. 0.043 ± 0.024, P = 0.045). CONCLUSIONS: Considering that this was a short-term study, because lenvatinib could be an agent that aggravates portal hypertension, it will be necessary to verify its clinical effects for portal hypertension in future studies.

Evoked potential studies for predicting functional recovery in a case of acute necrotizing encephalopathy.

Normal-appearing evoked potentials during the acute stage of the disease despite persistent coma may predict subsequent functional recovery of the brain in a pediatric case of acute necrotizing encephalopathy, indicating that evoked potential studies are useful for predicting functional outcome of the brain.

A very rare case of primary meningococcal arthritis in an adult male.

We report here a very rare case of primary meningococcal arthritis of the knee joint without clinical features associated with meningococcemia, meningitis, or meningococcal complications. The patient suffered from diabetes mellitus and had experienced two episodes of joint trauma. Intravenous infusion of ampicillin/sulbactam for 18 consecutive days was successful.

Impact of platelet transfusion on survival of patients with intracerebral hemorrhage after administration of anti-platelet agents at a tertiary emergency center.

This study examined the impact of platelet transfusion (PLT) on the survival of intracerebral hemorrhage (ICH) patients who had been administered anti-platelet agents (APA). This retrospective cohort analysis investigated 432 patients (259 men, 60%) who were newly diagnosed with ICH between January 2006 and June 2011 at the tertiary emergency center of Kitasato University Hospital. Median age on arrival was 67.0 years (range, 40-95 years). ICH was subcortical in 72 patients (16.7%), supratentorial in 233 (53.9%), and infratentorial in 133 (30.8%). PLT was performed in 16 patients (3.7%). Within 90 days after admission to the center, 178 patients (41.2%) had died due to ICH. Before the onset of ICH, 66 patients had been prescribed APA because of atherosclerotic diseases. Multivariate regression analysis indicated APA administration was an independent risk factor for death within 7 days (odds ratio, 5.12; P = 0.006) and within 90 days (hazard ratio, 1.87; P = 0.006) after arrival. Regarding the effect of a PLT in ICH patients with APA, no patient with PLT died. PLT had a survival benefit on patients with ICH, according to our analysis. Further prospective analysis is necessary to confirm the effects of PLT on survival in ICH with APA.

Is there a relation between triglyceride concentrations in very low density lipoprotein and the index of insulin resistance in nondiabetic subjects?

BACKGROUND: Serum very low density lipoprotein (VLDL) levels increase during the early stages of insulin resistance; therefore, determination of VLDL levels would be useful for evaluating the progression of metabolic syndrome and diabetes mellitus. The aim of this study was to clarify the clinical utility of triglyceride in VLDL (VLDL-TG) level, determined using a homogeneous assay kit (Shino-test Corporation, Tokyo, Japan), as an index of insulin resistance. METHODS: We enrolled 74 subjects in this study (diabetic subjects, n = 42; nondiabetic subjects, n = 32). The levels of VLDL-TG, remnant-like lipoprotein particle cholesterol, preheparin lipoprotein lipase mass, and other biochemical markers were determined. RESULTS: VLDL-TG levels were significantly higher in the diabetic group (1.04 ± 0.84 mmol/l vs. 0.64 ± 0.42 mmol/l, P < 0.01) than in the nondiabetic group. In the nondiabetic group, VLDL-TG was significantly correlated with the homeostasis model assessment of insulin resistance (HOMA-IR), the index for insulin resistance (r = 0.513, P = 0.003). VLDL-TG levels, but not TG levels, were higher in the highest quartile (HOMA-IR) of the nondiabetic group. CONCLUSION: VLDL-TG level was a useful early marker for insulin resistance, especially in nondiabetic subjects. The homogeneous VLDL-TG assay is a simple, low-cost method for determining insulin resistance.

[Clostridium tetani isolated from patients with systemic tetanus].

Clostridium tetani is widely distributed in ground or mud, especially in field and pond-shore surface layers. C. tetani is rarely isolated from specimens of patients with tetanus, and is generally diagnosed based on clinical symptoms such as trismus or general tonic spasms. This means that positive C. tetani infection is rarely diagnosed bacterially. Using gram straing, we identified C. tetani in specimens from patients suspected of C. tetani infection brought to the Kitasato University Hospital emergency center. Rapid gram staining information in the bacteriology laboratory is expected to improve recovery from C. tetani infection. It is therefore necessary to ensure clinical specimen quality control, and to keep standard strains of rare bacteria for isolation and identification.

Epidemiology of visceral mycoses in autopsy cases in Japan: the data from 1989 to 2009 in the Annual of Pathological Autopsy Cases in Japan.

To identify recent trends in the frequency of mycoses in autopsy cases, we analyzed, on a four-year basis, the 1989-2009 data in the Annual of Pathological Autopsy Cases in Japan. Of the 13,787 (9235 males) autopsies conducted in 2009, 4.5% (633/13,787) involved fungal infections and of the latter, 60.3% (368/633) were found to have severe clinical manifestations. Among the 610 (96.4%) cases involving a single etiologic angent, the predominant pathogens were Aspergillus (299 cases; 49%) and Candida (184 cases; 30.2%). However, it should be noted that the prevalence of severe aspergillosis and candidiasis has been decreasing. Although the frequency of cases involving zygomycetes seemed to be generally remaining stable from 1989-2009, we noted for the first time a peak in 2009 in such infections in patients less than one year old. Finally, deep-seated infections caused by unidentified fungi would appear to be decreasing over the time of the survey. Our finding, it is hoped, will encourage physicians to actively pursue viscerial fungal infections.

Progression of non-small cell lung cancer: diagnostic and prognostic utility of matrix metalloproteinase-2, C-reactive protein and serum amyloid A.

Matrix metalloproteinase-2 (MMP-2) is known to degrade type IV collagen, which is a major component of the cellular basement membrane, and to be involved in the invasion and metastasis of cancer cells. On the other hand, C-reactive protein (CRP) and serum amyloid A (SAA) are acute inflammatory biomarkers that increase in various conditions including infection, inflammation, malignancy and tissue disturbance. In the present study, we examined the serum levels of MMP-2, CRP and SAA in patients with localized and metastatic non-small cell lung cancer (NSCLC) to establish the clinical significance and changes in these biomarkers during NSCLC progression. In this study, 24 NSCLC patients were diagnosed at the Kitasato University Hospital and compared with 13 healthy controls. Measurement of MMP-2 levels in serum was determined by measuring pro-MMP-2 using a one-step sandwich enzyme immunoassay. CRP and SAA levels in the serum were measured by latex nephelometry. The serum levels of MMP-2, CRP and SAA in metastatic NSCLC patients were significantly higher than in localized NSCLC patients (p<0.01). There was a significant positive correlation between serum MMP-2 and CRP levels as well as SAA levels in metastatic NSCLC patients (p<0.01). Therefore, quantitation of MMP-2, CRP and SAA in NSCLC patients may be an auxiliary indicator to monitor tumor progression and poor prognosis of NSCLC disease.

[Emergence of linezolid-resistant Enterococcus faecalis strains from two inpatients in a pediatric ward].

We report herein on the isolation of three linezolid-resistant Enterococcus faecalis strains in 2011 from two pediatric inpatients at Kitasato University Hospital, Japan. Three linezolid resistant strains were isolated from two patients who shared the same room of a pediatric inpatient ward. Two linezolid resistant strains were isolated from patient A who had been treated with a total of 17,600mg of linezolid during 60 days of hospitalization (strains 1 and 2). The linezolid resistant E. faecalis persisted through the time that the patient had been discharged from the hospital. Another linezolid resistant strain was isolated from patient B who had no history of linezolid administration. The resistant strain in patient B phased out spontaneously. The minimum inhibitory concentration of linezolid in these strains ranged from 8.0 to 16.0 microg/mL. PCR amplification of the chromosomal gene encoding domain V of the 23S rRNA and subsequent nucleotide sequencing revealed that all the strains had at least one G2576T mutation. The pulse-field-gel electrophoretograms of the DNA treated with the SmaI restriction enzyme showed an identical profile suggesting that they were derived from a single resistant strain. These results suggested that the resistant strain occurred in patient A and was transmitted to patient B within the inpatient ward.

Changes of proteases and proteinase inhibitors in androgen-dependent advanced prostate cancer patients with alpha2-macroglobulin deficiency.

BACKGROUND: It is thought that the quantitative imbalance between proteases and their inhibitors is a causative factor in invasion and metastasis of cancer cells. We previously reported on a number of androgen-dependent advanced prostate cancer (PCa) patients in which serum alpha2-macroglobulin (alpha2M) levels were markedly decreased to < 20 mg/dL (defined as alpha2M deficiency). Anti-androgen therapy is at first generally very effective for androgen-dependent advanced PCa, yielding survival benefits for most patients. In the present study, we evaluated serum levels of PSA, matrix metalloproteinases-2 (MMP-2), alpha2M, and alpha2-plasmin inhibitor (alpha2PI) in advanced PCa patients with or without alpha2M deficiency in order to determine the clinical significance of these proteases and proteinase inhibitors for PCa progression. METHODS: In this study, 33 PCa patients were diagnosed at the Kitasato University Hospital and compared with 10 healthy controls. PSA and MMP-2 levels were determined by enzyme immunoassay. Measurement of alpha2M was performed by laser-nephelometry, alpha2PI levels were determined by turbidimetric immunoassay. RESULTS: Serum levels of PSA and MMP-2 in PCa patients with alpha2M deficiency were significantly higher than in patients not alpha2M-deficient. In contrast, serum levels of alpha2M and alpha2PI in these patients were significantly lower than in those not alpha2M-deficient. PSA and alpha2M levels showed an inverse relationship in androgen-dependent advanced PCa with alpha2M deficiency. CONCLUSIONS: Our findings indicate that the serum levels of these proteases and proteinase inhibitors, which are involved in the invasion and metastasis of PCa, may be indicators of PCa disease progression in addition to PSA levels.

Clinicopathological characteristics of androgen-dependent advanced prostate cancer patients with α2-macroglobulin deficiency.

α2-Macroglobulin (α2M) is thought to be involved in cancer metastasis and inflammatory reaction through its functions as a proteinase inhibitor and carrier protein for interleukin-6 (IL-6). We previously reported that advanced prostate cancer (PCa) patients with multiple distant bone metastases had markedly decreased serum α2M levels (<20 mg/dl) and no detection of α2M by immunoelectrophoresis (defined as α2M deficiency). We also showed a relationship between serum α2M levels and acute inflammatory biomarkers in PCa patients with or without α2M deficiency. In this study, we analyzed in detail the clinicopathological characteristics and pathogenesis of α2M deficiency in androgen-dependent advanced PCa patients. In this study, 15 PCa patients were diagnosed at the Kitasato University Hospital. α2M levels were determined by laser-nephelometry and immunoelectrophoresis, and PSA levels were determined by enzyme immunoassay. IL-6 levels were measured by a specific luminescence sandwich-type enzyme-linked immunosorbent assay, and CRP levels were determined by latex nephelometry. Immunohistochemical staining for PSA in PCa specimens was also performed. The binding assay for purified α2M and PSA was analyzed by western blotting. α2M deficiency was specific for advanced PCa patients with multiple distant bone metastases. PSA was markedly detected in sera and prostate specimens of advanced PCa patients with α2M deficiency, and there was a negative correlation between serum α2M and PSA levels during the course of clinical treatment. Acute inflammatory biomarkers such as IL-6 and CRP were within reference range in α2M-deficient patients. The binding assay showed that PSA easily bound to α2M, which was detected as an approximately 800-kDa complex by western blotting. Further, genetic analysis of a α2M-deficient patient showed no mutations in the α2M gene. These results suggested that α2M deficiency develops from catabolism of α2M in androgen-dependent advanced PCa patients, and serum α2M level may be an indicator of PCa disease progression in addition to PSA level.

Clinical usefulness of multiplex PCR lateral flow in MRSA detection: a novel, rapid genetic testing method.

Methicillin-resistant Staphylococcus aureus (MRSA) with exogenous cassette DNA containing the methicillin-resistant gene mecA (SCCmec) poses a problem as a drug-resistant bacterium responsible for hospital- and community-acquired infections. The frequency of MRSA detection has recently been increasing rapidly in Japan, and SCCmec has also been classified more diversely into types I-V. A rapid test is essential for early diagnosis and treatment of MRSA infections, but detection by conventional methods requires at least two days. The newly developed multiplex PCR lateral flow method allows specific amplification of femA to detect S. aureus, mecA to detect SCCmec, and kdpC to detect SCCmec type II; moreover, PCR products can be evaluated visually in about 3 h. In the present study, we developed a PCR lateral flow method for MRSA using this method and investigated its clinical usefulness in the detection of MRSA. The results showed a diagnostic concordance rate of 91.7% for MRSA and methicillin-susceptible S. aureus between bacteriological examination and PCR lateral flow, and a high level of specificity in PCR lateral flow. In addition, a higher detection rate for S. aureus using the same sample was observed for PCR lateral flow (70.2%) than for bacteriological tests (48.6%). The above results show that PCR lateral flow for MRSA detection has high sensitivity, specificity, and speed, and its clinical application as a method for early diagnosis of MRSA infections appears to be feasible.

Levels of acute inflammatory biomarkers in advanced prostate cancer patients with α2-macroglobulin deficiency.

C-reactive protein (CRP), serum amyloid A (SAA), interleukin-6 (IL-6), α1-antitrypsin (α1AT), α1-acid glycoprotein (α1AG) and ceruloplasmin (CP) are acute inflammatory biomarkers that increase in various conditions including infection, inflammation, malignancy and tissue disturbance. In contrast, α2-macroglobulin (α2M) is involved in inflammation through its function as a carrier protein of IL-6. We had previously reported on advanced prostate cancer (PCa) patients with multiple distant bone metastases in whom serum α2M levels were markedly decreased (α2M deficiency). However, the relationship between serum levels of α2M and acute inflammatory biomarkers in PCa patients with or without α2M deficiency has not been demonstrated. In the present study, we examined serum levels of CRP, SAA, IL-6, α1AT, α1AG and CP in PCa patients with or without α2M deficiency to establish clinical significance and changes in these biomarkers during PCa disease progression. We found that upon addition of recombinant IL-6 (rIL-6) to serum from PCa patients with α2M deficiency, since a function of α2M is to bind and stabilize IL-6, the α2M-IL-6 complex and free endogenous IL-6 were not detectable. Serum levels of the α2M-independent markers, α1AT, α1AG and CP, in all PCa patients regardless of α2M deficiency were significantly higher than in healthy controls, but those of the α2M-dependent molecules, CRP, SAA and IL-6, were not increased in PCa patients with α2M deficiency. Therefore, quantitation of both α2M-dependent (CRP, SAA and IL-6) and α2M-independent (α1AT, α1AG and CP) acute inflammatory biomarkers in advanced PCa patients may be an auxiliary indicator, together with prostate-specific antigen (PSA), to monitor PCa disease progression.

Acute inflammatory biomarkers in cerebrospinal fluid as indicators of blood cerebrospinal fluid barrier damage in Japanese subjects with infectious meningitis.

BACKGROUND: The blood-cerebrospinal fluid barrier (BCB) has selectivity for protein components with different molecular weights. Protein components in the cerebrospinal fluid (CSF) change when the BCB is damaged. We calculated the alpha2 macroglobulin (alpha2M) index as an indicator of BCB permeability from the point of view of molecular weight and evaluated the relationship between the alpha2M index and CSF concentrations of the inflammatory biomarkers interleukin-6 (IL-6), C-reactive protein (CRP), and serum amyloid A (SAA) in Japanese subjects with infectious meningitis, in order to determine the clinical significance of those inflammatory biomarkers in CSF. METHODS: IL-6, CRP, and SAA levels in CSF and serum were measured using various methods. The alpha2M index was calculated as the ratio of alpha2M (CSF/serum) to albumin (CSF/serum). RESULTS: CSF IL-6 levels were higher than serum IL-6 levels in 16 patients with infectious meningitis. The difference in CSF IL-6 and CRP levels between mycotic or bacterial meningitis cases and healthy controls and in CSF SAA levels between all infectious meningitis cases and healthy controls were significant. There was a significant positive correlation between CSF levels of CRP or SAA and alpha2M indices. CONCLUSIONS: Markedly increased levels of IL-6 in the CSF of patients with infectious meningitis may reflect the degree of intrathecal inflammation. On the other hand, increased CSF levels of CRP in patients with infectious meningitis, particularly mycotic or bacterial meningitis, and SAA in patients with all infectious meningitis may reflect the degree of damage to the BCB.

[Biochemical markers and physiological tests of atherosclerosis--changes and usefulness of markers in anti-atherosclerotic therapy].

As a result of the aging of the Japanese population and westernization of the diet, atherosclerotic diseases such as ischemic heart diseases and cerebrovascular disorders are now the leading causes (2nd and 3rd, respectively) of death in Japan. Furthermore, advances in medical technology have made Japanese some of the longest-lived citizens in the world, and increasing health care costs have become an object of public concern. Therefore, the prevention and treatment of atherosclerosis that causes these ischemic organ dysfunctions is indispensable. In Japan, diagnostic criteria were established for metabolic syndrome as one of the health care cost-containment policies, and the importance of controlling the blood lipid and glucose levels and blood pressure was defined in 2005. Furthermore, persons enrolled in the health insurance system aged between 40 and 74 years have been obliged to receive a specified health check for the prevention of metabolic syndrome since April 2008. The health check includes: a medical interview; anthropometry (height, body weight, BMI, and abdominal circumference); physical examination; blood pressure; and 8 laboratory tests (neutral fat, HDL-cholesterol, LDL-cholesterol, AST, ALT, gamma-GT, fasting blood glucose [or HbAlc], and urinary protein and glucose). In fact, the specified health check is becoming one of the enlightening activities concerning the maintenance and promotion of health for staff of Kitasato University Hospital. Recently, on the other hand, the mechanism in which inflammatory reactions are involved in atherosclerosis has been elucidated, and atherosclerosis is thought to be a chronic inflammatory disease. Furthermore, the advances in laboratory test methods have made the measurement of various sensitive markers for inflammation possible. In this presentation, we would like to explain the efficient application of biochemical markers and rapid physiological tests for the pathological diagnosis of atherosclerosis. We would also like to explain changes in anti-thrombotic therapy aiming at the primary prevention of atherosclerosis and its usefulness.

[Electroencephalographic background activity and blood biochemistry data in patients with chronic renal failure during chronic hemodialysis].

There is no particular electroencephalographic activity known to be associated with consciousness disturbance in uremic patients; however, a slow wave activity is generally observed during consciousness disturbance. Abnormal electroencephalographic activity was observed in 30 (67%) of 45 chronic renal failure patients during chronic hemodialysis without consciousness disturbance, and slow wave activity was observed in 58%. The frequency of the electroencephalographic background activity correlated with blood urea nitrogen (BUN) and serum Ca levels, but not with K, IP, and creatinine levels. Electroencephalographic activity can be estimated with reference to BUN or serum Ca levels in the blood of uremic patients.