Development and Evaluation of a Robust Sandwich Immunoassay System Detecting Serum WFA-Reactive IgA1 for Diagnosis of IgA Nephropathy.

Aberrant glycosylation of IgA1 is involved in the development of IgA nephropathy (IgAN). There are many reports of IgAN markers focusing on the glycoform of IgA1. None have been clinically applied as a routine test. In this study, we established an automated sandwich immunoassay system for detecting aberrant glycosylated IgA1, using Wisteria floribunda agglutinin (WFA) and anti-IgA1 monoclonal antibody. The diagnostic performance as an IgAN marker was evaluated. The usefulness of WFA for immunoassays was investigated by lectin microarray. A reliable standard for quantitative immunoassay measurements was designed by modifying a purified IgA1 substrate. A validation study using multiple serum specimens was performed using the established WFA-antibody sandwich automated immunoassay. Lectin microarray results showed that WFA specifically recognized N-glycans of agglutinated IgA1 in IgAN patients. The constructed IgA1 standard exhibited a wide dynamic range and high reactivity. In the validation study, serum WFA-reactive IgA1 (WFA+-IgA1) differed significantly between healthy control subjects and IgAN patients. The findings indicate that WFA is a suitable lectin that specifically targets abnormal agglutinated IgA1 in serum. We also describe an automated immunoassay system for detecting WFA+-IgA1, focusing on N-glycans.

Clinical evaluation of a new creatine kinase MB activity reagent abrogating the effect of mitochondrial creatine kinase.

BACKGROUND: Measuring creatine kinase (CK) MB activity using the immunoinhibition method remains useful in clinical laboratories. CK-MB activity is abnormally high when macro CK type 2 (mitochondrial creatine kinase, MtCK) is present in patient serum. In order to improve the accuracy of the CK-MB activity assay, we developed a new CK-MB activity method using highly specific anti-MtCK antibodies. We evaluated the clinical performance of the new method, which abrogates the effect of MtCK activity. METHODS: Receiver operating characteristic analysis, CK-MB activity range, cut-off, and CK-MB to CK activity ratio were investigated. RESULTS: Mean CK-MB activity in normal human serum was 2.5 U/L by our method, in contrast to 12.0 U/L by the current method. Approximately 80% of CK-MB activity determined using the current kit corresponds to MtCK activity, and ubiquitous mitochondrial creatine kinase activity constitutes approximately 90% of MtCK activity. The cut-off and CK-MB activity ratio of our method were 12 U/L and 3 to 20%, respectively, in contrast to 22 U/L and 5 to 23%, respectively, using the current CK-MB method. The areas under the curve of our method, current CK-MB, electrophoresis, and CK-MB mass were 0.976, 0.928, 0.967, and 0.991, respectively. Our new method was superior to the electrophoresis and CK-MB mass as well as the conventional method due to jts promptness, simplification, and low cost. CONCLUSIONS: The new kit will improve the clinical diagnosis of acute myocardial infarction. CK-MB activity assay was considered as a suitable alternative to conventional cardiac markers due to its superior diagnostic validity.

Preparation of highly purified monomeric human serum albumin as secondary reference material for standardization of urinary albumin immunoassays.

BACKGROUND: International external quality assessments have shown variation in results of urinary albumin among various immunoassays. A well-defined candidate reference material for urine albumin (cRM-UA) was prepared to improve standardization. METHODS: cRM-UA was prepared from a commercially available preparation of human serum albumin by using gel-filtration HPLC. The value was assigned by transfer from ERM-DA470 using immunoassay systems qualified based on the linearity and variability observed in dilution tests of pooled urine and the calibrators. Effectiveness of recalibration using the cRM-UA was evaluated by measuring 129 urine specimens. RESULTS: The cRM-UA had a monomeric albumin peak which accounted for 98.9% of the total area by gel filtration HPLC. The lyophilized preparation of the cRM-UA had suitable homogeneity, and short- and long-term stability. Nine of 14 immunoassays met the criteria were used for value assignment. The assigned concentration was 225.1±9.11 mg/l [mean±U: expanded uncertainty with k=2] when reconstituted with 3.00 ml of purified water on weight basis. Recalibration of 7 qualified immunoassays using the cRM-UA resulted in between-method CV of 6.6%. CONCLUSIONS: The cRM-UA was successful in achieving standardization of urine albumin results among 7 immunoassays which possess performance attributes representing uniform reactivity to both cRM-UA and clinical urine samples.

[Clinical evaluation of the new creatine kinase MB reagent kit "L-System CK-MB MtO"].

The creatine kinase MB (CK-MB) activity assay, which is based on the immunoinhibition method, has long been used in the diagnosis of acute myocardial infarction (AMI) because of its good cost-performance ratio and simplicity. However, the immunoinhibition method can not differentiate between CK-MB and MtCK, and therefore, CK-MB activity determined using this method is higher than the actual value in the sample which MtCK appears; this may lead to the misdiagnosis of AMI. We, therefore, evaluated the analytical and clinical performance of a new CK-MB reagent kit "L-System CK-MB MtO," which can inhibit MtCK. The kit yielded good precision and linearity and no interference from hemolysis, bilirubin or chyle. A good correlation was observed between the values determined using this kit and those determined using the conventional kit for samples of patients with acute coronary syndromes. However, differences were observed in the CK-MB values determined for samples from patients with malignancy. CK isoenzyme analysis indicated that MtCK was present in all these samples. The new method permits the accurate estimation of CK-MB activity in samples of patients with high serum levels of MtCK activity and indicates that the conventional method has a high false-positive rate for CK-MB activity. CK-MB activity in the serum of healthy individuals measured using the new and the conventional kits was 1.9-9.5 U/l and 4.5-15.3 U/l, respectively. The new kit, enables accurate estimation of CK-MB activity and is, therefore, more useful than the conventional kit in the diagnosis of acute coronary syndromes.

Development and performance of an enzyme immunoassay to detect creatine kinase isoenzyme MB activity using anti-mitochondrial creatine kinase monoclonal antibodies.

OBJECTIVE: The MB fraction of creatine kinase (CK-MB) has long been used as a cardiac marker. It is known that the CK-MB immunoinhibition method lacks selectivity and accuracy, because the appearance of macro CK type 2, corresponding to mitochondrial creatine kinase (MtCK) in some patient serum may render CK-MB activity measured by conventional method abnormally high. Thus, to improve the specificity and accuracy of the CK-MB assay, we developed two types of monoclonal anti-MtCK antibodies against sarcomeric MtCK and ubiquitous MtCK, and present herein the performance of a new method using these antibodies. MATERIAL AND METHODS: The performance of our test for detecting CK-MB activity was compared with other methods, and the range of CK-MB activities in normal human serum was investigated. RESULTS: The two types of monoclonal antibodies developed by us were isoenzyme-specific to sMtCK or uMtCK. The correlation coefficients of our method and conventional method to electrophoresis were 0.973 and 0.873, respectively. The mean CK-MB activity in normal human serum by our method and the conventional method was 2.4 and 11.7 U/L, respectively. Thus, our data indicated that about 80% of CK-MB activity, determined using the conventional method, seems to correspond to the MtCK activity. CONCLUSION: Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.

[Clinical significance of urinary cholesterol/total protein ratio as a marker of selectivity of proteinuria: comparison with selectivity index].

Selectivity index (SI) has been reported to reflect the selectivity of proteinuria, and it has a relationship with tubulointerstitial damage. Moreover it has a predictive value on functional outcome. However, it is necessary to measure serum IgG, serum transferrin, urinary IgG, and urinary transferrin to calculate SI. We measured urinary micro-cholesterol (mCHO) levels in sixty-three patients with proteinuria (urinary total protein > or = g/gCr) and compared urinary mCHO/total protein(uTP) ratio and SI. The patients' diseases were minimal change nephrotic syndrome (MCNS, n = 12), focal and segmental glomerular sclerosis (FSGS, n = 12), membranous nephropathy (MN, n = 17), and diabetic nephropathy (DMN, n = 22). Urinary mCHO levels were measured by the ECC method using cholesterol ester hydrolase (CEH) and cholesterol dehydrogenase, and this method was performed conveniently by automatic analyzer. No correlation was observed between urinary mCHO/gCr and serum lipid levels. There was no difference of urinary protein levels among each disease group. We found urinary mCHO/uTP ratio has a good positive correlation with SI(R = 0.722, p < 0.001). Although the difference between ROC curves of SI and urinary mCHO/uTP ratio in distinguishing MCNS from other diseases (FSGS+MN+DMN) did not reach the statistical significance, the area under the curve was larger for mCHO/uTP ratio. These results suggest that measurement of urinary mCHO by ECC method can be a simple and useful tool for predicting selectivity of proteinuria and lipoprotein-loading tubulopathy.

[A case of posterior mediastinal mature teratoma].

The chest radiograph of a 57-year-old man, complaining of paroxysmal dyspnea, suggested the probably of a tumor. Chest CT showed a tumor containing calcification, behind the left crus of the diaphragm. Chest MRI suggested lipid components and a cystic lesion within the tumor. Their findings were clinically compatible with posterior mediastinal teratoma. The pathological diagnosis of the surgically resected tumor was mature teratoma with neither malignant components nor thymic tissue. Study of past case reports suggests that posterior mediastinal teratomas should have less malignant characteristics than anterior mediastainal teratomas. Our case is the fifteenth case report in the Japanese literature, and accumulation of more cases is required.

[A case of occult thyroid cancer detected as a solitary nodular lung metastasis].

An 82-year-old woman was admitted to our hospital with cough and back pain. A chest radiograph showed a solitary nodular lesion in the right lower lung field. It was diagnosed by a transbronchial biopsy as lung metastasis of a papillary adenocarcinoma of the thyroid. However, her cervical CT and ultrasonography showed only a cyst in a right lobe of the thyroid, and its biopsy did not show evidence of malignancy. In addition, multiple bone metastasis and pituitary metastasis were revealed. We therefore diagnosed this case as systemic metastasis of papillary adenocarcinoma of the thyroid. She was given best supportive care and she died seven months later. Autopsy revealed two tiny lesions (3mm and 6mm) in the thyroid right lobe to be papillary adenocarcinoma. We report this case because occult thyroid cancer caused systemic metastasis and the chest X-ray showed lung metastasis from the thyroid cancer as a solitary nodular lesion.

[A case of AIDS-complicated lung infection mimicking acute eosinophilic pneumonia].

A 34-year-old man was admitted with dyspnea and low grade fever. Chest radiograph and computed tomography (CT) showed bilateral, ground glass opacities and perihilar consolidation. Bronchoalveolar lavage (BAL) was performed. The percentage of eosinophils in the BAL fluid (BALF) was elevated (20.5%). BALF smear and culture showed normal flora. Acute eosinophilic pneumonia was diagnosed and steroid therapy was performed. Afterwards he was transferred to our hospital. The HIV antibody was positive and peripheral blood CD-4 positive lymphocytes decreased to 10/microl, cytomegalovirus (CMV) antigenemia was positive and beta-D-glucan increased. CMV infection and pneumocystis pneumonia (PCP) complicated with AIDS was diagnosed. Trimethoprim/sulfamethoxazole, ganciclovir, and antifungal drugs were administered, but he suffered pneumothorax on the 18th day after admission and died. Histopathologic findings from an autopsy lung specimen revealed CMV infection and PCP. It is known that the percentage of eosinophils in the BALF increases in some cases of PCP complicated with AIDS. We emphasize that it is necessary to consider PCP when the percentage of eosinophils in the BALF increase.

Intralobar pulmonary sequestration presenting increased serum CA19-9 and CA125.

A 39-year-old man was admitted to our hospital for further evaluation of a consolidated shadow and clarification of the cause of serum tumor marker elevation (CA19-9 496.2 U/ml, CA125 160.6 U/ml). Chest computed tomography revealed a well-defined homogeneous nodule in the left S(10). Angiography showed one aberrant artery, branching from the ascending aorta. Intralobar pulmonary sequestration was diagnosed and the sequestrated lung was resected. Microscopic findings of the sequestrated lung showed a mucus-containing cystically dilated bronchus, which was covered with ciliated cylindrical epithelium. Immunohistochemical staining showed positive staining for CA19-9 and CA125 in both the ciliated cylindrical epithelium and mucus. Serum values of tumor markers returned to their normal range after surgery.