A Case of Waldenstrom Macroglobulinemia with Temporary Appearance of 7S IgM Half Molecule.

BACKGROUND: We encountered a rare case of Waldenstrom macroglobulinemia with temporary appearance of 7S IgM half molecule and with monoclonal proteins binding to agarose gel. METHODS: The patient's serum and urine were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The N-terminal amino acid sequences of the IgM with abnormal mass (68 kDa) were determined and compared with those of known immunoglobulin. RESULTS: The 68 kDa IgM consisted of a defective µ chain (36 kDa) and an intact κ chain. N-terminal amino acid sequence analysis demonstrated that the defective µ chain had the variable region of IgM. The agarose gel-binding ability of the IgM-κ M-protein was lost after reduction or alkaline treatment of serum. CONCLUSIONS: The 7S half molecule IgM in the present case may miss a large part of the constant region of the µ chain.

Difference in bias approach for commutability assessment: application to frozen pools of human serum measured by 8 direct methods for HDL and LDL cholesterol.

BACKGROUND: We used a difference in bias approach to evaluate the commutability of 4 frozen serum pools for 8 direct methods for measurement of HDL and LDL cholesterol (HDLC and LDLC). METHODS: Freshly collected nonfrozen sera from 138 diseased and 37 nondiseased patients and 4 frozen pools from the CDC Lipid Standardization Program were measured by direct methods and by the beta-quantification reference measurement procedure of the CDC. We used an error components model to estimate the difference in the bias component of error plus its uncertainty for frozen pools vs patient samples between the direct method and the reference procedure. Frozen pools with bias differences less than a critical value determined by either medical requirements for bias or the random error components of the measurement procedures were considered commutable. RESULTS: On the basis of medical requirement criteria, 1 of the 4 frozen pools was commutable for most of the HDLC methods for both diseased and nondiseased patients, and none was commutable for LDLC methods. On the basis of random error criteria, all of the frozen pools were generally commutable for all of the HDLC methods for both diseased and nondiseased patients, and 1 of the 4 frozen pools was generally commutable for most of the LDLC methods for both diseased and nondiseased patients. CONCLUSIONS: Commutability was assessed as the closeness of agreement of the difference in bias between a reference material and a set of patient samples. Criteria for commutability could be based on fixed medical requirements for bias or on random error components.

Non-HDL cholesterol shows improved accuracy for cardiovascular risk score classification compared to direct or calculated LDL cholesterol in a dyslipidemic population.

BACKGROUND: Our objective was to evaluate the accuracy of cardiovascular disease (CVD) risk score classification by direct LDL cholesterol (dLDL-C), calculated LDL cholesterol (cLDL-C), and non-HDL cholesterol (non-HDL-C) compared to classification by reference measurement procedures (RMPs) performed at the CDC. METHODS: We examined 175 individuals, including 138 with CVD or conditions that may affect LDL-C measurement. dLDL-C measurements were performed using Denka, Kyowa, Sekisui, Serotec, Sysmex, UMA, and Wako reagents. cLDL-C was calculated by the Friedewald equation, using each manufacturer's direct HDL-C assay measurements, and total cholesterol and triglyceride measurements by Roche and Siemens (Advia) assays, respectively. RESULTS: For participants with triglycerides<2.26 mmol/L (<200 mg/dL), the overall misclassification rate for the CVD risk score ranged from 5% to 17% for cLDL-C methods and 8% to 26% for dLDL-C methods when compared to the RMP. Only Wako dLDL-C had fewer misclassifications than its corresponding cLDL-C method (8% vs 17%; P<0.05). Non-HDL-C assays misclassified fewer patients than dLDL-C for 4 of 8 methods (P<0.05). For participants with triglycerides≥2.26 mmol/L (≥200 mg/dL) and<4.52 mmol/L (<400 mg/dL), dLDL-C methods, in general, performed better than cLDL-C methods, and non-HDL-C methods showed better correspondence to the RMP for CVD risk score than either dLDL-C or cLDL-C methods. CONCLUSIONS: Except for hypertriglyceridemic individuals, 7 of 8 dLDL-C methods failed to show improved CVD risk score classification over the corresponding cLDL-C methods. Non-HDL-C showed overall the best concordance with the RMP for CVD risk score classification of both normal and hypertriglyceridemic individuals.

The relationship between oxidized lipoprotein(a) and carotid atherosclerosis in asymptomatic subjects: a comparison with native lipoprotein(a).

BACKGROUND: Oxidized lipoprotein(a) (oxLp(a)) can be a more potent marker of atherogenesis than native Lp(a), although Lp(a) is considered to be a risk factor for atherosclerotic diseases. Limited clinical data are available regarding the significance of oxLp(a) in atherosclerotic manifestations. This study aimed to investigate the association between the serum oxLp(a) and carotid artery intima-media thickness (CIMT), in comparison to the serum Lp(a) levels, among asymptomatic subjects. METHODS: The atheroscrerosis-related variables including Lp(a) and oxLp(a) were measured in 136 cardiovascular disease-free subjects (61 males and 75 females, mean age of 64 years). The serum oxLp(a) level was quantified using a sandwich ELISA system. The CIMT level was ultrasonographically measured on bilateral carotid arteries. RESULTS: The median level of Lp(a) was 120 µmol/L, oxLp(a) was 0.06 nmol/L, and CIMT was 0.7 mm, respectively. A simple correlation test showed that the CIMT was significantly and positively correlated with age, systolic blood pressure and oxLp(a) (r = 0.208, P < 0.05). A multiple linear regression analysis revealed that oxLp(a) continued to show a significant and positive correlation with the CIMT (ß = 0.202, P = 0.01). Although the similar analyses were conducted for Lp(a), it showed only a weak correlation with the CIMT (r = 0.011, ß = 0.041, both P < 0.05). CONCLUSIONS: These results suggest that oxLp(a) may be more closely associated with accelerated carotid atherosclerosis, in comparison to Lp(a), in this population. This finding can be important for obtaining a better understanding of the different atherogenic roles played by oxLp(a) in comparison to Lp(a).

Seven direct methods for measuring HDL and LDL cholesterol compared with ultracentrifugation reference measurement procedures.

BACKGROUND: Methods from 7 manufacturers and 1 distributor for directly measuring HDL cholesterol (C) and LDL-C were evaluated for imprecision, trueness, total error, and specificity in nonfrozen serum samples. METHODS: We performed each direct method according to the manufacturer's instructions, using a Roche/Hitachi 917 analyzer, and compared the results with those obtained with reference measurement procedures for HDL-C and LDL-C. Imprecision was estimated for 35 runs performed with frozen pooled serum specimens and triplicate measurements on each individual sample. Sera from 37 individuals without disease and 138 with disease (primarily dyslipidemic and cardiovascular) were measured by each method. Trueness and total error were evaluated from the difference between the direct methods and reference measurement procedures. Specificity was evaluated from the dispersion in differences observed. RESULTS: Imprecision data based on 4 frozen serum pools showed total CVs <3.7% for HDL-C and <4.4% for LDL-C. Bias for the nondiseased group ranged from -5.4% to 4.8% for HDL-C and from -6.8% to 1.1% for LDL-C, and for the diseased group from -8.6% to 8.8% for HDL-C and from -11.8% to 4.1% for LDL-C. Total error for the nondiseased group ranged from -13.4% to 13.6% for HDL-C and from -13.3% to 13.5% for LDL-C, and for the diseased group from -19.8% to 36.3% for HDL-C and from -26.6% to 31.9% for LDL-C. CONCLUSIONS: Six of 8 HDL-C and 5 of 8 LDL-C direct methods met the National Cholesterol Education Program total error goals for nondiseased individuals. All the methods failed to meet these goals for diseased individuals, however, because of lack of specificity toward abnormal lipoproteins.

The association between blood glucose and oxidized lipoprotein(a) in healthy young women.

BACKGROUND: Oxidized lipoproteins play important roles in the atherosclerotic processes. Oxidized lipoprotein(a) (oxLp(a)) may be more potent in atherosclerotic pathophysiology than native Lp(a), a cardiovascular disease-relevant lipoprotein. Increased blood glucose concentrations can induce oxidative modification of lipoproteins. The aim of this study was to investigate the association between circulating oxLp(a) and cardiometabolic variables including blood glucose in healthy volunteers within the normal range of blood glucose. METHODS: Several cardiometabolic variables and serum oxLp(a) (using an ELISA system) were measured among 70 healthy females (mean age, 22 years). RESULTS: Lp(a) and glucose were significantly and positively correlated with oxLp(a) in simple correlation test. Furthermore, a multiple linear regression analysis showed oxLp(a) to have a weakly, but significantly positive and independent correlation with only blood glucose (ß = 0.269, P < 0.05). CONCLUSIONS: These results suggest that increased glucose may enhance the oxidization of Lp(a) even at normal glucose levels.

Novel automated pulse immunoassay for human alpha-fetoprotein.

BACKGROUND: To improve current alpha-fetoprotein (AFP) assays, which are expensive and time-consuming, a specific AFP reagent has been developed for practical use in our newly developed high-speed, highly sensitive pulse immunoassay (PIA) system, in which a latex immunoagglutination reaction is carried out under a high-frequency pulse voltage, leading to an enhanced immunological reaction. METHODS: We evaluated the assay performance (reproducibility, sensitivity, dilution linearity, interference) of the newly developed automated AFP PIA compared with the current AFP assay. RESULTS: Using pooled serum samples, the within-run reproducibility resulted in a correlation variation of 3.6-4.7%. The AFP assay detection limit was determined to be 2.5 microg/L. Linear sequential dilution was found up to nearly 700 microg/L. Even up to an AFP concentration of 1.0 g/L, the prozone phenomenon was not observed. Free and conjugated bilirubin, haemolytic haemoglobin, chyle and rheumatoid factor did not show any test interference. Using AFP-positive serum samples from 114 patients, the correlation between our PIA and a chemiluminescence immunoassay resulted in an excellent correlation coefficient of 0.994. CONCLUSIONS: The performance of AFP reagents in the PIA device shows that the system has excellent speed and equal sensitivity and specificity compared with the most highly sensitive conventional method. Our PIA system thus appears ready for use in the clinical diagnosis setting.

Serodiagnosis of Mycobacterium avium-complex pulmonary disease using an enzyme immunoassay kit.

RATIONALE: The diagnosis of Mycobacterium avium-complex pulmonary disease (MAC-PD) and/or its discrimination from pulmonary tuberculosis (TB) is sometimes complicated and time consuming. OBJECTIVES: We investigated in a six-institution multicenter study whether a serologic test based on an enzyme immunoassay (EIA) kit was useful for diagnosing MAC-PD and for distinguishing it from other lung diseases. METHODS: An EIA kit detecting serum IgA antibody to glycopeptidolipid core antigen specific for MAC was developed. Antibody levels were measured in sera from 70 patients with MAC-PD, 18 with MAC contamination, 37 with pulmonary TB, 45 with other lung diseases, and 76 healthy subjects. MEASUREMENTS AND MAIN RESULTS: Significantly higher serum IgA antibody levels were detected in patients with MAC-PD than in the other groups (P < 0.0001). Setting the cutoff point at 0.7 U/ml resulted in a sensitivity and specificity of the kit for diagnosing MAC-PD of 84.3 and 100%, respectively. Significantly higher antibody levels were also found in patients with nodular-bronchiectatic disease compared with fibrocavitary disease in MAC-PD (P < 0.05). There was a positive correlation between the extent of disease on chest computed tomography scans and the levels of antibody (r = 0.43, P < 0.05) in patients with MAC-PD. CONCLUSIONS: The EIA kit is useful for the rapid diagnosis of MAC-PD and for differentiating MAC-PD from pulmonary TB and, if validated by studies in other populations, could find wide application in clinical practice.

Higher frequency of abnormal serum angiopoietin-like protein 3 than abnormal cholesteryl ester transfer protein in Japanese hyperalphalipoproteinemic subjects.

BACKGROUND: Either a decrease of cholesteryl ester transfer protein (CETP) or an increase of angiopoietin-like protein 3 (ANGPTL3) in plasma has been shown to increase HDL-cholesterol (HDL-C) levels. However, as yet, it is not known which protein is more strongly associated with the modulation of HDL in the Japanese hyperalphalipoproteinemic (HALT) subjects. METHODS: The serum concentration of ANGPTL3 and CETP, together with total cholesterol (TC), triglycerides (TG), adiponectin and ApoE phenotypes were determined in three groups with different HDL-C concentrations: low, <40 mg/dl (n=51); normal, 40-90 mg/dl (n=126) and high, >90 mg/dl (n=89) in the average Japanese population. RESULTS: The normal range (mean+/-2SD) of serum ANGPTL3 (218+/-144 ng/ml) and CETP (1.29+/-0.90 microg/ml) were determined in cases with 40-90 mg/dl HDL-C concentration. The frequency of abnormally high ANGPTL3 cases (>362 ng/ml) were found to be significantly greater (44%) compared with those of low CETP cases (<0.39 microg/ml, 4.5%) in HALT cases (>90 mg/dl). ANGPTL3 showed a high correlation with HDL-C (r=0.67, P<0.0001) and adiponectin (r=0.57, P<0.0001), but not with CETP. CONCLUSION: In average Japanese population, abnormally higher frequency of increased ANGPTL3 prevail in HALT cases as compared with cases with low CETP. These findings suggest that ANGPTL3, the inhibitor of endothelial lipase, may be more strongly associated with increased HDL-C rather than CETP in plasma. Accordingly, ANGPTL3 seems to be a better target for the modulation of HDL-C.

Innovative instrumental analysis of heartbeat signals and its clinical application.

A new apparatus has been built that annexes a normal electrocardiograph, ECG, with the aim to enhance its capacity. It adds the normal ECG power to superpose multiple records of ECG altogether and averages out them and, further, makes sophisticated analysis, such as normalizing the peak heights, evaluating the half-line widths of the peaks, or that of the standard deviations of measurements like the inter peak distances. The results of the said calculations have not been obtainable using the former instruments, and are expected to be useful for clinicians.

[Evoked potential monitoring in an operation of neurosurgery].

In the neurosurgical field, the evoked potential is employed for the monitoring of intraoperative nerve function. During evoked potential monitoring, surgical manipulation-related nerve dysfunction is detected, and functional localization/nerves in the cerebral cortex are identified to prevent postoperative neurological complications. It is important to reduce the contact resistance to 2 kOmega or less on the application of plate electrodes used for recording and prevent noise by bundling electrode leads, to ensure a stable evoked potential during surgery. In our laboratory, intraoperative monitoring, such as ABR to prevent auditory disturbance, SEP to detect cerebral ischemia, cortical SEP and MEP to prevent motor paralysis, and evoked electromyography to identify/maintain the cranial nerves including the facial, trigeminal, oculomotor, and abducens nerves, is performed based on requests from the Department of Neurosurgery.

The development of an artificial stool usable for the surveillance of faecal haemoglobin testing.

Background Faecal occult blood testing is an important diagnostic tool for the detection of colorectal cancer. However, it has not been standardized due to the absence of suitable specimens for surveillance. Methods We developed a ready-to-use artificial stool made from rice flour. This new artificial stool homogeneously contains not only human haemoglobin A0 (HbA0) but also glycerol as an internal standard material. After the collection of the artificial stool into a buffer, the haemoglobin concentration in dispersed solution was measured using a method based on the peroxidase like activity of haemoglobin. The glycerol concentration was measured using a commercially available triglyceride measurement kit. Results With regard to the haemoglobin stability, the decrease in the level of human haemoglobin in the artificial stool was <2% when it was stored at -80℃ for four months, -20℃ for two weeks, and 5℃ for two days. The artificial stool was easily collected with the collecting tubes of a commercially available faecal haemoglobin test kit. The weight of the collected artificial stool could be calculated by measuring the concentration of glycerol in the extracting solution of the collected stool sample. The haemoglobin concentrations could be adjusted based on their collection weights. Conclusions The artificial stool has a paste-like consistency and contains both haemoglobin and glycerol homogeneously. Furthermore, the measured haemoglobin concentration could be determined based on the collected stool weight, which was directly related to the glycerol concentration. These features make it a useful material for the surveillance of faecal occult blood testing.

Novel artificial stool material for external quality assurance (EQA) on a fecal immunochemical test for hemoglobin (FIT): The confirmed utility of stable hemoglobin and an internal standard material.

The fecal immunochemical test for hemoglobin (FIT), which detects lower gastrointestinal bleeding, is widely accepted for population-based colorectal cancer (CRC) screening programs. However, the FIT screening process has not been standardized yet, and standardizing the pre-analytical phase and establishing an external quality assurance (EQA) program compliant with ISO requirements is urgently needed. Although there have been various attempts to establish EQA materials suitable for FIT, no materials have yet been reported to have sufficient uniformity and acceptable immunochemical stability of hemoglobin (Hb). The Health Care Technology Foundation (HECTEF; Tokyo Japan) is now developing a ready-to-use artificial stool containing Hb and an internal standard, glycerol. Accordingly, we verified the adaptability and efficacy of this material for the evaluation of the specimen collection phase of FIT. This material uniformly contained both Hb and glycerol. The glycerol allowed us to estimate the weight of the collected artificial stool and to correct the Hb concentration with the estimated weight. Furthermore, the stability of both Hb and glycerol were confirmed to be sufficient for an EQA material under appropriate storage, in-use, repeated freeze-thaw, and heated conditions. These in-house performance characteristics suggest that HECTEF artificial stool is acceptable as an EQA material for FIT.

Serum remnant lipoprotein cholesterol/triglyceride ratio as an index for screening familial type III hyperlipidaemia.

BACKGROUND: An elevated serum remnant lipoprotein cholesterol (RLP-C)/triglyceride (TG) ratio has not been evaluated as an index of familial type III hyperlipidaemia defined by the presence of beta-VLDL and apolipoprotein (Apo) E2/2 phenotype in the Japanese hyperlipidaemic population. METHODS: Serum lipids and lipoproteins from 514 individuals (200 men and 314 women, mean age 58 years) with total cholesterol >6.22 mmol/L and TG between 2.26 mmol/L and 9.04 mmol/L, selected from 25,080 subjects visiting the clinics for health checkup were analysed for a possible relationship with familial type III hyperlipoproteinaemia. RESULTS: Median RLP-C concentration and RLP-C/TG ratio were 0.30 and 0.11 mmol/L, respectively. When compared between subjects with (31 cases) and without (483 cases) a broad-beta band on electrophoresis, the RLP-C concentrations and RLP-C/TG ratio were 0.77 +/- 0.43 mmol/L versus 0.34 +/- 0.16 mmol/L (P<0.0001) and 0.15 +/- 0.023 versus 0.11 +/- 0.027 (P<0.0001), respectively. Three cases with broad-beta band positive (the presence of beta-VLDL) showed RLP-C/TG ratio greater than 0.23 and RLP-C greater than 0.78 mmol/L, suggestive of type III hyperlipoproteinaemia, despite a lack of characteristic Apo E2/2 homozygosity. Cases with Apo E/Apo CIII ratio greater than 1.0 were not detected in this study group. CONCLUSION: Serum RLP-C concentration and RLP-C/TG ratio, together with Apo E/Apo CIII ratio appear to be useful for screening familial type III hyperlipidaemia in the Japanese hyperlipidaemic population.

Proposal of automation of candidate reference method for the accurate serum cholesterol assay in clinical laboratories.

BACKGROUND: In 2001, Japan Society of Clinical Chemistry (JSCC) recommended the cholesterol dehydrogenase (CD)-UV method as a comparative method for serum cholesterol measurement in Japan. Although the CD-UV method is intended to standardize routine laboratory tests, it requires complex manipulations, and has been difficult to use for the evaluation of clinical laboratories. We therefore attempted to automate this method using reagents specified by JSCC and developed a simple automated method. METHODS: We evaluated the simple automated method using 2 general instruments (JCA-BM12 and H-7170S). The linearity was confirmed for the range over 15.52 mmol/l. The coefficients of variation for 20 measurements of serum containing 2.5 and 6.1 mmol/l of cholesterol were < 1.0%, respectively. No interference by bilirubin, ditauro bilirubin, hemoglobin and chylomicrons was observed in this method. When measurement data with JCA-BM12 were compared with those using the comparative method, the correlation coefficient was 0.999 (n=23), the regression formula was y=0.992x - 0.0036 (mmol/l), and the bias was 0.8%. A similar data was obtained with H-7170S. Thus, in both comparisons, the bias was within the target (+/- 3.0%). CONCLUSIONS: This automated method provides a valid means of implementing the serum cholesterol measuring method recommended by JSCC.

An immunoglobulin A1 that inhibits lactate dehydrogenase activity, with reversal of inhibition by addition of NADH.

We discovered a patient with low serum lactate dehydrogenase (LD) activity and an abnormal LD isozyme pattern. We analyzed the patient's LD inhibitor using electrophoresis, affinity chromatography, and immunochemical technologies. The LD activity of the patient's serum was inhibited more strongly at 4 degrees C than at 37 degrees C. The decrease of LD activity was more marked in a mixture of the patient's serum with purified LD5 than in that with purified LD1. The immunoglobulin responsible for LD inhibition was an IgA1-lambda. The LD inhibition by the patient's IgA1 was blocked by reduction and alkylation and by NADH. Polymerization of the patient's IgA1 might play an important role in its interaction with LD. Moreover, the possibility exists that part of the patient's IgA1 molecule fits into a pocket of LD in instead of NADH. This is the first report of NADH reversing such LD inhibition.

[Evaluation of samples that indicated the difference of immunoglobulin values between different assays].

Since the immunoglobulin levels of survey samples for the survey of Japanese Association of Medical Tech7ologists in 2003, especially the IgA and IgM, became different by the immunological methods (LAIA [latex agglutination immunoassay] and TIA [turbidimetric immunoassay]), the cause of difference was investigated. From the results of gel filtration and immunoblotting analysis, it was suggested that profiles of the reaction of purified IgA and IgM polymers in survey samples were different on each method. In the case of M protein, also, there were higher levels of IgM and IgA type M protein by TIA and SRID methods than other methods even though the precipitation curve was obscure. Similarly, several samples from patients with M protein demonstrated higher values by SRID method than that of other methods. When these samples were analyzed by gel filtration and immunoblotting, they did not indicate the presence of the polymeric IgM. It will be suggested that non-specific reactions on TIA (weak reaction) and SRID (unusual precipitin line) methods were present. Furthermore, in a case of IgM type M protein, IgM level was twice higher by LAIA than that by TIA. However, it did not indicate any polymer or fragment of IgM by gel filtration and immunoblotting. Accordingly, it was assumed that the difference in measured values between the methods was attributable to the difference of the reaction to the samples.

[Current status for OTC examination (test)].

Upgrading and expanding of preventive measures for lifestyle-related diseases is an urgent national priority because they are recognized as the most important health risks by both the general population and government. With such a social background, the spread of self-medication by self-test is eagerly awaited by health-oriented individuals. In this paper, legal position of OTC test reagent, OTC test equipment and remote healthcare as current status of OTC test are explained, reference is made to the current status of their diffusion here in Japan, and some typical examples of "self-collection sample and corresponding OTC test item" are clearly described. In consideration of the large number of potential patients with folk disease such as metabolic syndrome, drug stores should be an accessible and convenient spot for Japanese people to enjoy the benefit of OTC test by obtaining test kits for such diseases.

[Future outlook for OTC examination (test)].

As the primary prevention of disease has been emphasized, there may be a growing need for well-conditioned development and spread of OTC test equipment, OTC test reagent and remote healthcare. Some typical examples of "simple and rapid examination items, which can be done at home by established technology" are explained in this paper and may be required in the near future by the whole Japanese population with a view to the current status of OTC test and the coming relaxation of governmental regulation. The reasons why these examination items should be commercialized as Switch OTC and the epidemiological background are further discussed. In relation to metabolic syndrome derived from super spreader-like visceral mature adipocyte: SSVMAC, visceral fat rate measurement by bioelectrical impedance technology, postprandial blood glucose determination/urinary glucose determination, and urinary microalbumin detection, are explained with an example, together with the importance of early morning blood pressure recording. In the field of cancer and infectious diseases, immunological fecal occult blood determination as a marker of distal colorectal cancer, in which both higher survival rate and recovery rate by early detection have been established, Helicobacter pylori IgG antibodies or antigen examination including an examination of gastroduodenal disorder, prostate-specific antigen (PSA) and genital chlamydia are emphasized.