Serum amyloid A does not affect high-density lipoprotein cholesterol measurement by a homogeneous assay.

BACKGROUND: Serum amyloid A (SAA), which is one of the acute phase proteins, alters the structure of HDL by associating with it during circulation. We focused on whether SAA influences the values of HDL-cholesterol (HDL-C) measurements when using a homogeneous assay. METHODS: HDLs were isolated by ultracentrifugation from serum samples of 248 patients that were stratified into three groups based on their serum SAA concentrations (low: SAA ≤ 8 µg/mL; middle: 8 < SAA ≤ 100 µg/mL; and high: SAA > 100 µg/mL). HDL-C concentrations of the serum samples measured by the homogeneous assay were compared with the total cholesterol concentrations of HDL fractions isolated by ultracentrifugation. RESULTS: HDLs obtained from patients with low SAA concentrations were separated into their general particle sizes and classified as HDL2 and HDL3 by native-gel electrophoresis. On the other hand, HDLs obtained from patients with high SAA concentrations occasionally showed distributions different from the typical sizes of HDL2 and HDL3, such as extremely small or large particles. Nevertheless, HDL-C concentrations measured using the homogeneous assay were strongly correlated with those measured using the ultracentrifugation method, regardless of the SAA concentrations. However, the ratios of HDL-C concentrations obtained by the homogeneous assay to those obtained by the ultracentrifugation method for patients with high SAA concentrations were significantly lower than those of patients with low SAA concentrations. CONCLUSIONS: A large amount of SAA attached to HDL altered the HDL particle size but did not essentially affect HDL-C measurement by homogeneous assay.

Measurement of plasma choline in acute coronary syndrome: importance of suitable sampling conditions for this assay.

Blood choline has been proposed as a predictor of acute coronary syndrome (ACS), however different testing procedures might affect the choline concentration because the lysophospholipase D activity of autotaxin (ATX) can convert lysophosphatidylcholine to lysophosphatidic acid (LPA) and choline in human blood. Although the influences of ATX on LPA levels are well known in vivo and in vitro, those on choline have not been elucidated. Therefore, we established suitable sampling conditions and evaluated the usefulness of plasma choline concentrations as a biomarker for ACS. Serum LPA and choline concentrations dramatically increased after incubation depending on the presence of ATX, while their concentrations in plasma under several conditions were differently modulated. Plasma choline levels in genetically modified mice and healthy human subjects, however, were not influenced by the ATX level in vivo, while the plasma LPA concentrations were associated with ATX. With strict sample preparation, the plasma choline levels did not increase, but actually decreased in ACS patients. Our study revealed that ATX increased the choline concentrations after blood sampling but was not correlated with the choline concentrations in vivo; therefore, strict sample preparation will be necessary to investigate the possible use of choline as a biomarker.

Validation and application of a novel cholesterol efflux assay using immobilized liposomes as a substitute for cultured cells.

Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.

A simple, rapid and validated high-performance liquid chromatography method suitable for clinical measurements of human mercaptalbumin and non-mercaptalbumin.

Background Human mercaptalbumin and human non-mercaptalbumin have been reported as markers for various pathological conditions, such as kidney and liver diseases. These markers play important roles in redox regulations throughout the body. Despite the recognition of these markers in various pathophysiologic conditions, the measurements of human mercaptalbumin and non-mercaptalbumin have not been popular because of the technical complexity and long measurement time of conventional methods. Methods Based on previous reports, we explored the optimal analytical conditions for a high-performance liquid chromatography method using an anion-exchange column packed with a hydrophilic polyvinyl alcohol gel. The method was then validated using performance tests as well as measurements of various patients' serum samples. Results We successfully established a reliable high-performance liquid chromatography method with an analytical time of only 12 min per test. The repeatability (within-day variability) and reproducibility (day-to-day variability) were 0.30% and 0.27% (CV), respectively. A very good correlation was obtained with the results of the conventional method. Conclusions A practical method for the clinical measurement of human mercaptalbumin and non-mercaptalbumin was established. This high-performance liquid chromatography method is expected to be a powerful tool enabling the expansion of clinical usefulness and ensuring the elucidation of the roles of albumin in redox reactions throughout the human body.

Effects of serum amyloid A on the structure and antioxidant ability of high-density lipoprotein.

Serum amyloid A (SAA) levels increase during acute and chronic inflammation and are mainly associated with high-density lipoprotein (HDL). In the present study, we investigated the effect of SAA on the composition, surface charge, particle size and antioxidant ability of HDL using recombinant human SAA (rhSAA) and HDL samples from patients with inflammation. We confirmed that rhSAA bound to HDL3 and released apolipoprotein A-I (apoA-I) from HDL without an apparent change in particle size. Forty-one patients were stratified into three groups based on serum SAA concentrations: Low (SAA ≤ 8 µg/ml), Middle (8 < SAA ≤ 100 µg/ml) and High (SAA > 100 µg/ml). The ratios of apoA-I to total protein mass, relative cholesterol content and negative charge of HDL samples obtained from patients with high SAA levels were lower than that for samples from patients with low SAA levels. Various particle sizes of HDL were observed in three groups regardless of serum SAA levels. Antioxidant ability of rhSAA, evaluated as the effect on the formation of conjugated diene in low-density lipoprotein (LDL) induced by oxidation using copper sulfate, was higher than that of apoA-I. Consistent with this result, reconstituted SAA-containing HDL (SAA-HDL) indicated higher antioxidant ability compared with normal HDL. Furthermore, HDL samples obtained from High SAA group patients also showed the highest antioxidant ability among the three groups. Consequently, SAA affects the composition and surface charge of HDL by displacement of apoA-I and enhances its antioxidant ability.

Novel round cells in urine sediment and their clinical implications.

BACKGROUND: Voided urine contains a variety of cells from the kidney and urinary tract and urinalysis provides us various information by investigating cellular components. We investigated urine sediment from renal impaired patients. RESULTS: We found 'round cell' to be distinct from known cells in sediment and is close to proximal convoluted tubule-derived cells based on morphology and molecular marker expression (GGT1 but not podocalyxin). Also it was positive for undifferentiated cell markers, including PAX2, WT1, OSR1, and SIX2. They were observed in end-stage renal failure patients and the number of cells was correlated with the severity of chronic kidney disease. A prospective analysis revealed that patients who had more round cells were more likely to require hemodialysis within a year. CONCLUSION: Round cells are a novel marker that can be used to predict the need for hemodialysis.

[Evaluation of Immature Platelet Fraction Measurements Using an Automated Hematology Analyzer, Sysmex XN].

The immature platelet fraction (IPF%) is a parameter for the automatic quantification of reticulated plate- lets and is considered to reflect platelet production. We evaluated IPF% measurements using an automated analyzer, the Sysmex XN. We measured the platelet counts and the IPF% values in 35 healthy subjects and 275 patients with various diseases using both the XN analyzer and a conventional analyzer, the Sysmex XE. Significant correlations in the platelet count and the IPF% were observed between the XN results and the XE results. In the same samples, significant inverse correlations were observed between the platelet count and the IPF% in both the XN and XE results. The coefficient of variation values for the platelet count and the IPF% measurements obtained using the XN analyzer were lower than those obtained using the XE ana- lyzer. In patients who had undergone hematopoietic stem cell transplantation, the IPF% measurements obtained using the XN analyzer increased several days before platelet recovery. IPF% measurements performed using the XN analyzer are adequate for clinical use. This parameter may be a useful marker for the prediction of platelet recovery. [Original].

[IgM-Lambda Type Paraproteinemia Interferes with Haptoglobin Measurements].

Paraproteinemia is a condition induced by an increase in paraprotein. Paraprotein is a monoclonal immu- noglobulin produced by plasma cells as a result of aging or malignancy. Paraprotein often induces a variety of laboratory test abnormalities by interfering with laboratory test reagents. The haptoglobin (Hp) measurement in a 55-year-old woman with IgM-lambda type paraproteinemia associated with lymphoplasmacytic lymphoma was found to be falsely low because of white-turbidity caused by an abnormal reaction. An in- creased absorbance was observed at 596 nm after the addition of the buffer reagent and was reduced by dilution with saline. This result indicates the existence of interfering substances in the patient's sample. To identify the inhibitors, we obtained the white-turbidity pellet by centrifugation of a mixture of the patient's serum and the Hp buffer reagent. A high IgM concentration was observed in the white-turbidity pellet. Moreover, a correlation was observed in a time series between the IgM concentration in sera and the extent of the turbidity during the Hp measurement. These results indicated that IgM was the main component of the white-turbidity. Next, we showed that the addition of the white-turbidity pellet to normal serum caused an increase in absorbance and a false low Hp value. A correlation was also observed in a time series be- tween the IgM concentration in sera and the rate of the Hp reduction. These results suggest that the false low Hp measurements were due to IgM present in the white-turbidity. In conclusion, false low values of Hp may occur in patients with IgM-lambda type paraproteinemia. Therefore, the presence or absence of an increase in absorbance after the addition of the buffer reagent in a time-course reaction may be required.

[Harmonization of TSH Measurements.]

The measured concentration of thyroid stimulating hormone (TSH) differs depending on the reagents used. Harmonization of TSH is crucial because the decision limits are described in current clinical practice guide- lines as absolute values, e.g. 2.5 mIU/L in early pregnancy. In this study, we tried to harmonize the report- ed concentrations of TSH using the all-procedure trimmed mean. TSH was measured in 146 serum samples, with values ranging from 0.01 to 18.8 mIU/L, using 4 immunoassays. The concentration of TSH was highest with E test TOSOH and lowest with LUMIPULSE. The concentrations with each reagent were recalculated with the following formulas: E test TOSOH 0.855x-0.014; ECLusys 0.993x+0.079; ARCHITECT 1.041x- 0.010; and LUMIPULSE 1.096x-0.015. Recalculation eliminated the between-assay discrepancy. These formulas may be used until harmonization of TSH is achieved by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC).

[How to Treat the Clinical Specimens for Laboratory Testing in Line with Ethical Guidelines - Chairpersons' Remarks -].

Clinical laboratory studies utilizing residual specimens after laboratory testing have markedly contributed to the development of medical science. However, according to recent rules regarding ethical aspects, the ethical committee of the Japanese Society of Laboratory Medicine (JSLM) developed ethical guidelines for treating residual specimens. In this symposium, several ethical problems concerning the application of such specimens to laboratory investigation were raised and discussed. Concerning whether or not each informed consent should be ob- tained before the secondary use of the residual specimens, this matter itself revealed serious disagreements among institutes. Therefore, the ethical committee of JSLM has to propose uniform and widely acceptable guidelines for the effective utilization of laboratory specimens, aiming at the advancement of laboratory medi- cine. [Review].

Possible involvement of sphingomyelin in the regulation of the plasma sphingosine 1-phosphate level in human subjects.

OBJECTIVES: Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid mediator. Although the plasma S1P concentration is reportedly determined by cellular components, including erythrocytes, platelets, and vascular endothelial cells, the possible involvement of other factors, such as serum sphingomyelin (SM) and autotaxin (ATX), remains to be elucidated. DESIGN AND METHODS: We measured S1P using high-performance liquid chromatography (HPLC), SM and lysophosphatidic acid (LPA) using enzymatic assays, ATX antigen using a two-site enzyme immunoassay, and ATX activity using a lysophospholipase D activity assay. To fractionate the lipoproteins, plasma samples were separated using fast protein liquid chromatography (FPLC) utilizing a Superose 6 column. RESULTS: The plasma S1P level was positively correlated with the levels of SM and lysophosphatidylcholine, but not with the level of phosphatidylcholine. Although SM was present in the very low-density lipoprotein (VLDL) fraction, neither the plasma S1P level nor the SM level was affected by feeding. The plasma S1P level was negatively correlated with the ATX activity. Although the incubation of 100 µmol/L of sphingosylphosphorylcholine (SPC) with the serum resulted in a significant increase in the S1P level because of the presence of ATX, the physiological concentration of SPC did not mimic this effect. CONCLUSION: The plasma S1P level was affected by the serum SM level, while the possibility of ATX involvement in the increase in the plasma S1P level was considered to be remote at least in healthy human subjects.

Evaluation of the short-term stability of specimens for clinical laboratory testing.

BACKGROUND: A major concern in both the laboratory-medicine and research communities is the quality of human specimens for analysis. However, there is insufficient scientific evidence regarding optimal conditions for handling and storing routine specimens, especially those in liquid form. Thus, we investigated the stability of clinically relevant samples stored under various conditions. MATERIALS AND METHODS: Ten clinical laboratories in Japan conducted analyses of the stability of post-clinical (left over after analysis) test samples in relation to temperature and storage duration. We examined serum, whole blood, and urine samples submitted to each laboratory for routine testing. In this study, at least 5 samples for each of 35 tests were analyzed at each laboratory. After completion of routine testing, specimens with sufficient residual volume and values between LL-R/2 (lower limit of reference interval) and UL+R/2 (upper limit) were divided into 300 µL aliquots, where R=UL - LL. Aliquots of serum specimens were stored at either room temperature (23°C), 4°C, -20°C, or -80°C without light exposure. Aliquots of whole blood and urine specimens were stored at either 23°C or 4°C. The storage time was either 1, 3, or 7 days. Average differences between pre- and post-storage test results were evaluated for each laboratory test by two-way ANOVA. F-values for between-day variations were used for judging the statistical significance of storage-related changes in test values, whereas the ratio of between-day SD to between-individual SD (one-fourth of reference interval) was used to indicate the practical significance of the change. RESULTS AND CONCLUSION: Sample denaturation is clearly temperature- and storage-duration dependent for almost all analytes. In general, specimens were most susceptible to denaturation at 23°C, then 4°C, -20°C, and -80°C. This study confirmed the accumulated routine, practice-based, detailed knowledge regarding specimen stability and will help to ensure the reliability of laboratory test results.

Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells.

BACKGROUND: The migration of T cell to atherosclerotic lesions is proposed to be involved in the pathogenesis of the atherosclerosis. Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from activated platelets, exerts a variety of responses such as cell migration and proliferation, and reportedly induces T cell migration. Accordingly, platelet-T cell interactions may exist based on T cell responses triggered by platelet-derived S1P. METHODS: S1P was measured using two-step lipid extraction followed by high-performance liquid chromatography (HPLC) separation while other phospholipids were determined by an enzymatic assay. The expression of S1P and lysophosphatidic acid receptors on Jurkat T cells was examined by RT-PCR and flow cytometry. Jurkat cell migration by S1P and the supernatant of activated platelets (SAP) was evaluated by a modified Boyden's chamber assay. RESULTS: S1P1 receptor was confirmed to be expressed on Jurkat T cell by RT-PCR and flow cytometry. S1P at 10-100 nM induced strong Jurkat cell migration, which was inhibited by the S1P1 (and S1P3) antagonist VPC23019 and the Gi inactivator pertussis toxin (PTX). We found that the supernatant (releasate) of human platelets activated by collagen stimulation, which contains S1P abundantly, induced Jurkat cell migration and that the migration was inhibited by VPC23019 and PTX. In addition, human serum, into which platelet contents (including S1P) are fully released, induced the Jurkat cell migration, which was also inhibited by VPC23019. CONCLUSIONS: Our findings suggest that platelet-derived S1P induces Jurkat T cell migration possibly via S1P1. S1P may be a key molecule involved in the responses triggered by platelet-T cell interactions, including atherosclerosis.

[Performance and clinical evaluation of antinuclear antibody test based on fluorescence enzyme immunoassay].

BACKGROUND: The measurement of antinuclear antibodies (ANA) is used for screening of connective tissue diseases (CTD) in the laboratory. ANA detection is performed by indirect immunofluorescence (IF) assay on HEp-2 cells from human larynx carcinoma. However, it lacks specificity for the identification of specific diseases and antigen reactivity. The aim of the present study was to evaluate the EliA CTD Screen (EliA), a new enzyme fluoroimmunoassay (Phadia AB, Uppsala, Sweden) for detection of ANA in human serum. PATIENTS AND METHODS: The study involved a total of 732 serum samples, 200 from healthy donors, 297 from patients with CTD and 235 from patients with rheumatoid arthritis, vasculitis syndrome and relative disease of CTD. For all sera, ANA was measured by IF, commercial assay (MESACUP) and EliA. RESULT: The sensitivity and specificity of EliA were 73.7% and 78.7%, respectively, whereas those of MESACUP were 80.8% and 64.7%, respectively. Area under the receiver operating curves for EliA, MESACUP and IF were 0.821, 0.786 and 0.730, respectively. The concordance rate between EliA and MESACUP was 84.2%. These discrepancies between those 2 assays were found in 84 sera. Further investigation were done by each ANA antigen tests for the discrepant results of EliA in 83 sera. The discrepancies might be occurred by antigen difference or non-specific response. CONCLUSION: AUC results showed that the diagnostic performance of EliA was superior to MESACUP and IF. EliA had a good performance as method for screening of CTD.

Detection of novel visible-light region absorbance peaks in the urine after alkalization in patients with alkaptonuria.

BACKGROUND: Alkaptonuria, caused by a deficiency of homogentisate 1,2-dioxygenase, results in the accumulation of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) in the urine. Alkaptonuria is suspected when the urine changes color after it is left to stand at room temperature for several hours to days; oxidation of homogentisic acid to benzoquinone acetic acid underlies this color change, which is accelerated by the addition of alkali. In an attempt to develop a facile screening test for alkaptonuria, we added alkali to urine samples obtained from patients with alkaptonuria and measured the absorbance spectra in the visible light region. METHODS: We evaluated the characteristics of the absorption spectra of urine samples obtained from patients with alkaptonuria (n = 2) and compared them with those of urine specimens obtained from healthy volunteers (n = 5) and patients with phenylketonuria (n = 3), and also of synthetic homogentisic acid solution after alkalization. Alkalization of the urine samples and HGA solution was carried out by the addition of NaOH, KOH or NH4OH. The sample solutions were incubated at room temperature for 1 min, followed by measurement of the absorption spectra. RESULTS: Addition of alkali to alkaptonuric urine yielded characteristic absorption peaks at 406 nm and 430 nm; an identical result was obtained from HGA solution after alkalization. The absorbance values at both 406 nm and 430 nm increased in a time-dependent manner. In addition, the absorbance values at these peaks were greater in strongly alkaline samples (NaOH- KOH-added) as compared with those in weakly alkaline samples (NH4OH-added). In addition, the peaks disappeared following the addition of ascorbic acid to the samples. CONCLUSIONS: We found two characteristic peaks at 406 nm and 430 nm in both alkaptonuric urine and HGA solution after alkalization. This new quick and easy method may pave the way for the development of an easy method for the diagnosis of alkaptonuria.

[Difference in measurements of erythrocyte sedimentation rate obtained using two devices of the same model in a patient with marked leukocytosis].

The erythrocyte sedimentation rate (ESR) has been used as an index for inflammatory conditions, such as infectious diseases, autoimmune diseases, and malignancies. The ESR values of a 37-year-old male with marked leukocytosis due to chronic myeloid leukemia showed remarkable differences between two devices of the same model (Ves-Matic 30, DIESSE Diagnostica Senese). From the appearance of the tested tube after the ESR measurement, the values obtained using one device might have been falsely low, whereas the values obtained using the other device were likely to have been accurate. The difference of the ESR values between the two devices might have occurred by the false detection of transmitted light during the transition from the erythrocyte layer to the leukocyte layer. These findings suggest that in cases with marked leukocytosis the accuracy of ESR should be confirmed with the appearance of the test tube.

[Sphingolipids, possible biomarkers for atherosclerotic disorders].

Sphingolipids have been recently elucidated to be not only mere components of the plasma membrane but also bioactive mediators which can induce various biological responses. Among these lipids, sphingomyelin(SM) and sphingosine 1-phosphate (Sph-1-P) are proposed to be involved in the pathogenesis of atherosclerosis: SM is abundant in atherosclerotic lesions and Sph-1-P is bound to HDL and attributes to the anti-atherosclerotic properties of HDL at least partly. Therefore, Sph-1-P and SM can be useful biomarkers for atherosclerotic disorders. However, at present, the measurement of Sph-1-P and SM levels has not been brought into clinical practice, yet. The main obstacle is the difficulty in measuring these sphingolipids precisely, rapidly, and conveniently. Recently, we have developed new methods for measuring Sph-1-P (HPLC method) and SM (enzymatic method). These methods are easy to be introduced into clinical laboratory testing because they do not need any special techniques and equipment. With this method for SM, we have demonstrated that the SM concentration was significantly higher in subjects with acute coronary syndrome. In this paper, we reviewed the possibility of sphingolipids as biomarkers for atherosclerotic disorders.

Development of an enzymatic assay for sphingomyelin with rapid and automatable performances: Analysis in healthy subjects and coronary heart disease patients.

BACKGROUND: Sphingomyelin (SM) is an important choline group-containing phospholipid and is considered to be an independent risk factor for coronary heart disease. METHODS: We have developed a specific enzymatic assay for SM measurement with rapid and automatable performances by using two-reagent system involving sphingomyelinase. We performed within-run and between-run precision, linearity test, detection limit, recovery test and interference to validate this assay. Then, we measured the serum SM concentration in 194 healthy subjects and 141 consecutive patients undergoing coronary angiography. RESULTS: The within-run and between-run coefficients of variation for SM concentrations were 1.1-1.3% and 1.0-1.2%, respectively. Quantitative measurements to a lower limit of 30 µmol/L were shown to be possible. The recoveries of the exogenously added SM to the control samples were 98.7%-101.5%. No effect was observed after the addition of some interference materials. The mean ± SD of the serum SM concentration in the 194 healthy subjects was 553.3 ± 100.1 µmol/L. We found that the SM concentration was significantly higher among an acute coronary syndrome subjects than among the healthy subjects (P<0.01) and that the serum SM concentrations were significantly correlated with the serum magnesium concentration. CONCLUSIONS: We have developed a rapid and automatable enzymatic assay for SM that enables the automatic measurement of choline-containing phospholipids. This assay may be useful for various types of biochemical and clinical research.

[The performance of the automated immune chemiluminescent system "IMMULITE 2000XPi for the measurement of serum soluble IL-2 receptor in clinical samples].

The performance of a chemical luminescence test reagent "Immulyze IL-2R II" with an automated immune chemiluminescent system "IMMULITE 2000XPi" for the measurement of serum soluble IL-2 receptor in clinical samples was investigated. The satisfactory results were obtained for the reproducibility, precision, linearity, and sensitivity, and no interference with hemolysis, bilirubin, chyle or intrafat was observed. A significant correlation was found between the values of sIL-2R measured by the Cell-free N IL-2R and those obtained by the IMMULYZE IL-2R II. The measurements were stable regardless of the methods of sample preservation, or repeated freeze-thawing procedures. Elevated concentrations of sIL-2R over 1,000 U/mL were found in multiple types of collagen diseases or severe cases of allergic diseases, indicative that sIL-2R levels might correlate with the severity of autoimmune diseases. In patients with lymphoma, sIL-2R levels correlated with the lactate dehydrogenase (LD) activity. Among the lymphoma cases with sIL-2R levels over 1,000 U/mL, the majority (84%) had significantly higher levels of LD, and among them, 81% were at the clinical stage IV. We observed that sIL-2R levels increased from the early stages of lymphoma, while LD activities increased at the advanced stages. Our present findings suggest that sIL-2R is a promising marker for the diagnosis of autoimmune and allergic diseases, and also for the diagnosis and staging of lymphomas.