Showing papers in "Journal of Clinical Laboratory Analysis in 2008"

Evaluation of ischemia-modified albumin in anemia associated to chronic kidney disease.

Abstract: Chronic kidney disease (CKD) is highly prevalent, with increasing numbers of patients affected by the disease world-wide, and anemia is a common finding in patients with CKD. Anemia impacts negatively on cardiovascular disease, exercise capacity, and quality of life, resulting in significant mortality and morbidity. The aim of this study was to evaluate the levels of ischemia-modified albumin and lactate in patients with established anemia associated with CKD and its correlations with hemoglobin levels. Hematocrit, hemoglobin, iron, ferritin, albumin, creatinine, lactate, and ischemia-modified albumin (IMA) were measured in 17 patients with established anemia associated to CKD and 19 controls by standard methods. The results of hematocrit, hemoglobin, iron, and albumin were lower in the anemia group than in the control group. Ferritin, creatinine, and lactate levels were higher in anemia of the CKD group than the control group. IMA increase in the anemia group (0.8115±0.1304 absorbance units [ABSU]) compared to control (0.4951±0.0393 ABSU). Significant correlations between IMA and lactate, IMA and hemoglobin, IMA and creatinine, and hemoglobin and lactate were observed. IMA and lactate increase during anemia and this elevation could be associated to hypoxia due to low hemoglobin levels. However, our data suggest that lactate is more sensitive to anemia compared to IMA. J. Clin. Lab. Anal. 22:1–5, 2008. © 2008 Wiley-Liss, Inc.

Spread of nontuberculous mycobacteria from 1993 to 2006 in Koreans.

Abstract: In Korea, the prevalence of nontuberculous mycobacterial (NTM) pulmonary disease has risen, observed primarily in immunocompetent patients with or without preexisting lung disease. The purpose of this study was to determine the frequency of various species of NTM isolates from respiratory specimens in a single institution over a 14-year period in Korea. All samples referred to our reference laboratory over a 14-year period in Korea were analyzed. From 1993 to 2000 our laboratory used conventional NTM identification methods, and from 2001 we adapted PCR-restrictionfragment length polymorphism analysis(PRA). A total of 17,915 isolates were collected from 1993 to 2006. The most frequently isolated organisms were M. avium complex (n=11,705, 65%), M. abscessus (n=2,076, 11.59%), M. fortuitum complex (n=1,279, 7.14%). M. chelonae complex (n=1,134, 6.33%), M. kansasii (n=762, 4.25%), M. szulgai (n=139, 0.78%), M. celatum (n=87, 0.49%), M. scrofulaceum (n=18, 0.10%) and M. marium (n=11, 0.06%). J. Clin. Lab. Anal. 22:415–420, 2008. © 2008 Wiley-Liss, Inc.

Clinical laboratory automated urinalysis: comparison among automated microscopy, flow cytometry, two test strips analyzers, and manual microscopic examination of the urine sediments.

Abstract: Urinalysis is one of the habitual clinical laboratory procedures, which implies that one of the largest sample volumes currently requires significant labor to examine microscopic sediments. Different analyzers currently used to perform this task have been compared with the manual microscopic sediment examination. The Atlas Clinitek 10 (Bayer Corporation, Diagnostics Division, Tarrytown, NY) and Urisys 2400 (Hitachi Science Systems Ltd., Ibaraki, Japan) test strips analyzers and two automated urinalysis systems, Sysmex UF-100 (Sysmex Corporation Kobe, Japan) and IRIS iQ200 (International Imaging Remote Systems, Chatsworth, CA), have been considered. We assessed the concordance between the results obtained from 652 freshly collected urine samples for erythrocytes (RBC), leukocytes (WBC), squamous epithelial cells (EC), nitrites/bacteria, and crystals using the methodologies mentioned. A principal components analysis was performed in order to examine the correlation between these parameters. Instrument accuracy was also assessed. The Spearman's statistic (p) showed an adequate agreement between methods for RBC (iQ200=0.473; UF-100=0.439; Atlas=0.525; Urisys=0.539), WBC (iQ200=0.695; UF-100=0.761; Atlas=0.684: Urisys=0.620), and bacteria/nitrites (iQ200=0.538; UF-100=0.647; Atlas=0.532; Urisys=0.561) counts. By applying the Wilcoxon and McNemar tests, a concordance degree was found between 82-99 and 52-95% for the values obtained from the two test strips analyzers considered and from the iQ200 and UF-100 systems, respectively. From these results, we can conclude that both test strips analyzers are similar and, on the other hand, that automated urinalysis is needed to improve precision and the response time; but sometimes manual microscopic revisions are required, mainly when flags, because of crystals, are detected.

Nontreponemal tests in the diagnosis of neurosyphilis: an evaluation of the Venereal Disease Research Laboratory (VDRL) and the Rapid Plasma Reagin (RPR) tests

Abstract: The Venereal Disease Research Laboratory (VDRL) test has long been considered the best serological test for the diagnosis of neurosyphilis. The goal of this study was to find out if the Rapid Plasma Reagin (RPR) could be an alternative to the VDRL. Cerebrospinal fluid (CSF) and sera samples from patients in the following stages of syphilis were tested: 8 had symptomatic and 16 asymptomatic neurosyphilis, 4 were in the primary stage, 6 had secondary syphilis, and 92 were in the latent stage. We have also studied 61 samples from individuals with treated syphilis and 126 with other neurological diseases than neurosyphilis. All the CSF samples were studied with both RPR and VDRL tests. RPR and VDRL test results were mostly concordant. The specificity of these tests for current neurosyphilis was 99% for the VDRL and 99.3% for the RPR, whereas the sensitivity was 70.8 and 75%, respectively, for the VDRL and RPR. In view of these results it seems to us that the RPR could be an alternative to the VDRL in the diagnosis of neurosyphilis.

Clinical and laboratory evaluation of idiopathic male infertility in a secondary referral center in India.

Abstract: The genetic basis of infertility has received increasing recognition in recent years, particularly with the advent of assisted reproductive technology. It is now becoming obvious that genetic etiology for infertility is an important cause of disrupted spermatogenesis. Y-chromosome microdeletions and abnormal karyotype are the two major causes of altered spermatogenesis. To achieve biological fatherhood, intracytoplasmic sperm injection (ICSI) is performed in cases of severe infertility with or without genetic abnormalities. There is a concern that these genetic abnormalities can be transmitted to the male progeny, who may subsequently have a more severe phenotype of infertility. A total of 200 men were recruited for clinical examinations, spermiograms, hormonal profiles, and cytogenetic and Yq microdeletion profiles. Testicular biopsy was also performed whenever possible and histologically evaluated. Genetic abnormalities were seen in 7.1% of cases, of which 4.1% had chromosomal aberrations, namely Klinefelter's mosaic (47XXY) and Robertsonian translocation, and 3.0% had Yq microdeletions, which is very low as compared to other populations. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) were significantly increased in men with nonobstructive azoospermia (NOA) as compared to severe oligoasthenozoospermia (P<0.0001), whereas testosterone levels were significantly decreased in men with microdeletions as compared to men with no microdeletions (P<0.0083). Low levels of androgen in men with microdeletions indicate a need to follow-up for early andropause. Patients with microdeletions had more severe testicular histology as compared to subjects without deletions. Our studies showed a significant decrease (P<0.002) in the serum inhibin B values in men with NOA, whereas FSH was seen to be significantly higher as compared to men with severe oligoasthenozoospermia (SOAS), indicating that both the Sertoli cells as well the germ cells were significantly compromised in cases of NOA and partially affected in SOAS. Overall inhibin B in combination with serum FSH would thus be a better marker than serum FSH alone for impaired spermatogenesis. In view of the genetic and hormonal abnormalities in the group of infertile men with idiopathic severe oligozoospermia and NOA cases, who are potential candidates for ICSI, genetic testing for Y-chromosome microdeletions, karyotype, and biochemical parameters is advocated.

Anti-histones antibodies in systemic lupus erythematosus: prevalence and frequency in neuropsychiatric lupus.

Abstract: To investigate the specificity, sensitivity, and concomitant presence of antibodies against histones (H), histone H1 (H1), and histone H3 (H3) in patients with systemic lupus erythematosus (SLE) and analyze their association with SLE. Serum IgG anti-histones antibodies were detected by enzyme-linked immunosorbent assay in 144 SLE patients consisting of 24 neuropsychiatric lupus (NPSLE), 65 lupus nephritis (LN), and 55 SLE, 100 other rheumatic diseases patients, as well as 40 healthy controls. Clinical and biological parameters of the patients were also evaluated. Anti-H, anti-H1, and anti-H3 antibodies yielded a sensitivity of approximately 33% and a specificity of more than 93% for SLE, which was comparable to that found for anti-double-stranded DNA (anti-dsDNa) antibodies. More significantly, anti-histone antibody is found in approximately 50% of patients with NPSLE compared with LN. Moreover, the titers of anti-histones antibodies of NPSLE patients were significantly higher than that of patients with SLE and LN. The sequential analysis revealed a close correlation of anti-H and anti-H1 antibodies with SLE disease activity. There was an approximate 30% positive rate of anti-histones antibodies in 144 SLE patients lacking anti-nucleosome, anti-mDNA, anti-Sm, and anti-dsDNA antibodies. Antibodies to histones H1 and H3 are markers with high specificity of 93.6-96.4% for SLE. The anti-histone antibody markers are prevalent in approximately 50% of NPSLE. Furthermore, there was a strong correlation with SLE disease activity index and levels of antibodies to histones and H1.

Elevated serum level and gene polymorphisms of TGF-beta1 in gastric cancer.

Abstract: Transforming growth factor (TGF)-beta1, as a candidate tumor marker, is currently of interest. In this study, serum TGF-beta1 levels in gastric cancer (GC) patients and healthy volunteers were measured using enzyme-linked immunosorbent assay (ELISA). In addition, single nucleotide polymorphisms (SNPs) of the TGF-beta1 gene at codon 10 and codon 25 were identified by means of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and sequence analysis. Our results indicated that serum concentrations of TGF-beta1 in GC patients were significantly higher than those in the control, and positively correlated with tumor mass, invasion, metastasis, and clinical stage. The serum TGF-beta1 levels of patients recovering from radical resection were markedly lower than those before surgery. Meanwhile, no deoxyribonucleic acid (DNA) sequence variation at codon 25 of the TGF-beta1 gene was found and a TGF-beta1 gene polymorphism at codon 10 did not show obvious correlations with either TGF-beta1 expression or clinicopathological parameters of GC. Our evidence suggested that serum concentration of TGF-beta1 might be a novel tumor marker for GC and the polymorphisms of TGF-beta1 gene did not play a role as a determinant of serum TGF-beta1 concentration or as a genetic risk factor in the gastric carcinogenesis and progression.

Acute phase phospholipids related to the cardiolipin of mitochondria in the sera of patients with chronic fatigue syndrome (CFS), chronic ciguatera fish poisoning (CCFP), and other diseases attributed to chemicals, Gulf War, and marine toxins

Abstract: This study examined 328 CFS sera in a study with 17 CCFP, 8 Gulf War Veterans (GWV), 24 Prostate Cancer (PC), and 52 normal sera in the modified Membrane Immunobead Assay (MIA) procedure for CTX. Three hundred and twenty-eight CFS patients' sera were examined by the modified MIA with purified MAb-CTX and 91.2% gave a titre > or =1:40. 76% of the 17 CCFP sera samples and 100% of the 8 GWV sera samples also had a titre > or =1:40. 92.3% of 52 normal sera showed titres of 1:20 or less, while 4 gave titres of > or =1:40. In addition, 41 sera were examined for Anti-Cardiolipin (aCL) by a commercial ELISA procedure with 87.8% demonstrating IgM, IgM+IgA, or IgM+IgG aCL antibodies. These results showed mostly the IgM aCL antibody alone in the sera samples. In addition, 41 serum samples were examined for aCL, with 37 showing positive for aCL, representing 90.2% positive for the three disease categories examined: CFS, CCFP and GWV. Examination for antiMitochondrial-M2 autoantibody (aM-M2) in 28 patients (CFS (18), CCFP (5), and GWV (5)) was negative for aM-M2. Inhibition analysis with antigens, CTX, CFS "Acute Phase Lipids", commercial Cardiolipin (CL) and 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L-Serine] (PS) and antibodies, MAb-CTX and aCL from patients' serum show that the phospholipids in CL and CTX are antigenically indistinguishable with antibodies MAb-CTX and CFS-aCL. Preliminary chemical analyses have shown the lipids to be phospholipids associated with CL of the mitochondria. We designate this "Acute Phase Lipid" comparable to "Acute Phase Proteins" (C-reactive protein (CRP) and Serum Amyloid A (SAA)) in inflammatory conditions.

Alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) activity in the sera of patients with liver cancer

Abstract: The principal enzymes catalyzing the conversion of ethanol to acetate are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). The activities of these enzymes are elevated in the serum during the course of alcoholism or cirrhosis. In previous investigations we have found elevated levels of ADH, ALDH, and class I ADH activity in liver cancer cells. It can suggest that these changes may be reflected by enzyme activity in the serum. In this work, the activity of ADH isoenzymes, and ALDH in the sera of patients with liver cancer was measured. Serum samples were taken from 64 patients (28 drinkers, 36 nondrinkers), with liver cancer. 25 patients had primary and 39 metastatic liver tumors. Total ADH activity was measured by photometric method with p-nitrosodimethylaniline (NDMA) as a substrate and ALDH activity by the fluorimetric method with 6-methoxy-2-naphtaldehyde as a substrate. For the measurement of the activity of class I and II isoenzymes we employed the fluorimetric methods, with class-specific fluorogenic substrates. The activity of class III ADH was measured by the photometric method with formaldehyde and class IV with m-nitrobenzaldehyde as a substrate. A statistically significant increase of class I ADH isoenzymes was found in the sera of cancer patients. The median activity of this class isoenzyme in the total cancer group increased about 51% (2.94 mU/L) in the comparison to the control level (1.43 mU/L). The activity of the class I ADH isoenzyme was significantly higher in the sera of patients with metastatic tumors than with primary cancers. The activity of this class in the sera of drinkers and group of moderate drinkers was significantly higher in comparison to the control group and higher in the sera of heavy drinkers when compared with moderate drinking patients. The total ADH activity was significantly higher (44%) among patients with cancer than healthy ones. The activity of class I ADH isoenzymes was elevated only in the serum of patients with metastatic liver cancer. This increase of activity seems to be caused by the enzyme released from liver cancer cells and primary tumors originating in other organs.

Relationship between smoking and dyslipidemia in western Chinese elderly males

Abstract: The relationship between smoking and dyslipidemia was studied in 2,160 elderly Chinese males. Levels of triglycerides (TGs) in current smokers were shown to be significantly higher and levels of high-density lipoprotein cholesterol (HDL-C) lower than for those who had never smoked or had stopped smoking. Interestingly, the level of apoprotein B (apoB) was more frequently abnormal in very heavy smokers compared with light smokers, while low-density lipoprotein cholesterol (LDL-C) levels were more frequently normal in very heavy smokers. When comparing other factors that influence blood lipids, such as alcohol intake, body mass index (BMI), and age, smoking had the greatest influence and was shown to be an independent risk factor for dyslipidemia.

Prediction of diagnosis of immunoglobulin A nephropathy prior to renal biopsy and correlation with urinary sediment findings and prognostic grading.

Abstract: Several clinical markers correlate well with the diagnosis and prognosis of IgA nephropathy (IgAN). In the present study, we re-evaluated the usefulness of these four clinical markers for prediction of the diagnosis of patients with IgAN through a comparison between many more patients with IgAN and those with other types of renal diseases. 364 patients with IgAN and 289 with other types of renal disease were examined. An analysis was performed prior to renal biopsy, using clinical markers including, serum IgA, serum IgA/C3 ratio, number of red blood cells in urinary sediments, and urinary protein. Patients with IgAN were divided into four groups according to histopathological findings. Presence of microscopic hematuria, persistent proteinuria, high serum IgA levels, and the serum IgA/C3 ratios are useful for prediction of diagnosis of IgAN and distinguishing it from other renal diseases. Blood pressure, urinary protein, serum uric acid, renal function, and urinary sediment findings may be useful for prediction of prognostic grading in patients with IgAN.

Rapid saliva processing techniques for near real-time analysis of salivary steroids and protein.

Abstract: Introduction: Point-of-care (POC) measurements using saliva samples have immense potential to assess systemic health and wellbeing, but sample viscosity and contaminants can affect analyses. We sought a portable clean-up method for whole saliva appropriate for use with POC measurement techniques such as biosensors. Methods: Whole saliva from each of 13 male subjects was split into 5 fractions. Each fraction was treated with a different clean-up process: a freeze–thaw–centrifuge (FTC) step; centrifugation alone; or passage through a Mini-UniPrep polyethersulfone filter, cotton Salivette®, or foam Oracol device. Following clean-up, each subject's treated saliva fractions were assayed for cortisol, testosterone, dehydroepiandrosterone (DHEA), and proteinconcentrations. The effects of clean-upmethods on nonspecific binding (NSB) in a biosensor were also assessed. Results: Compared with FTC, no analytes were affected by centrifugation alone. Cotton Salivettes significantly altered all analytes, with increases in cortisol (+64%), testosterone (+126%), and DHEA (off-scale) levels, and decreased protein (−21%) and biosensor NSB (−75%). Oracol foam devices decreased DHEA levels by 28%. Mini-UniPrep filtration decreased testosterone (−45%) and DHEA (−66%) concentrations while increasing cortisol (+40%). Conclusion: No method was optimal for all analytes, highlighting the need for validation of saliva treatment methods before their adoption in rapid POC analyses. J. Clin. Lab. Anal. 22:395–402, 2008. © 2008 Wiley-Liss, Inc.

RNA from Borna disease virus in patients with schizophrenia, schizoaffective patients, and in their biological relatives.

Abstract: Numerous interactions of the immune system with the central nervous system have been described recently. Mood and psychotic disorders, such as severe depression and schizophrenia, are both heterogeneous disorders regarding clinical symptomatology, the acuity of symptoms, the clinical course, the treatment response, and probably also the etiology. Detection of p24 RNA from Borna disease virus (BDV) by the reverse transcriptase polymerase chain reaction in patients with schizophrenia, schizoaffective disorder, and in their biological relatives was evaluated. The subjects were 27 schizophrenic and schizoaffective patients, 27 healthy controls, 20 relatives without psychiatric disease, and 24 relatives with mood disorder, who attended the Psychiatric Ambulatory of Londrina State University, Parana, Brazil. The subjects were interviewed by structured diagnostic criteria categorized according to the Diagnostic and Statistical Manual of Mental Disorders-IV, axis I, (SCID-IV). The mean duration of illness in schizophrenic and schizoaffective patients was 15.341±1.494 years and the median age at onset was 22.4±7.371 years. There were no significant differences in gender (P=0.297), age (P=0.99), albumin (P=0.26), and body mass index (kg/m2) (p=0.28), among patients, controls, and relatives. Patients and biological relatives had significantly higher positive p24 RNA BDV detection than controls (P=0.04); however, the clinical significance of BDV remains to be clarified. J. Clin. Lab. Anal. 22:314–320, 2008. © 2008 Wiley-Liss, Inc.

Use of vaginal pH in diagnosis of infections and its association with reproductive manifestations

Abstract: Increase in vaginal secretion pH is an indicator of bacterial vaginosis (BV), but is yet to be in use as a diagnostic tool by clinicians. Similarly, no reports are available on the effect of cervical chlamydia infection and different reproductive manifestations on vaginal secretion pH. This study evaluated the use of vaginal pH for screening of BV, the effect of Chlamydia trachomatis (C. trachomatis) infection, and different reproductive manifestations on vaginal pH of women attending the gynecology outpatient department of a general hospital. Vaginal pH was recorded while diagnosing infections in 358 women, among which 45 were with repeated spontaneous abortion, 79 with infertility, 185 had sign and symptoms of lower genital tract infection, and 49 had no history or symptom of any complications or infections. Normal vaginal pH, BV, and C. trachomatis infection were observed in 72.6, 21.5, and 10.1% of women, respectively. BV and C. trachomatis were observed in 78.6 and 4.1% of women, respectively, with high vaginal pH; 12.3% of women with normal vaginal pH had C. trachomatis infection. C. trachomatis infection or different reproductive manifestations do not lead to change in vaginal pH but high vaginal pH correlated with BV and should be used as a simple tool for its diagnosis. J. Clin. Lab. Anal. 22:375–379, 2008. © 2008 Wiley-Liss, Inc.

Assay of ischemia-modified albumin and C-reactive protein for early diagnosis of acute coronary syndromes.

Abstract: Diagnosis of cardiac ischemia in patients coming to emergency departments (ED) with symptoms of acute chest pain is often difficult. Many markers are sensitive and specific for the detection of myocardial necrosis but may not rise during reversible myocardial ischemia. Ischemia-modified albumin (IMA) has recently been shown to be a sensitive and early biochemical marker of ischemia. The variation laws were observed by measuring IMA and C-reactive protein (CRP) of 113 patients in ED within 12 hr after onset of chest pain. In the observation, blood was taken for IMA and CRP. Patients underwent standardized triage, diagnostic procedures, and treatment. Results of IMA and CRP were correlated with final diagnoses of nonischemic chest pain (NICP) and acute coronary syndrome (ACS). There were obvious distinction of IMA and CRP levels between the NICP and ACS groups. Receiver operator characteristic (ROC) curve analysis was used to determine the optimal cutoff of this assay for identifying individuals with ACS patients from NICP. The area under the curves of IMA is 0.948. The sensitivity and specificity of albumin cobalt binding (ACB) at a cutoff value of 70.0 units/mL were 94.4% and 82.6%, respectively. The area under the curves of CRP is 0.746. Sensitivity and specificity of CRP at a cutoff value of 3.16 mg/L were 70.0% and 73.9%, respectively. Negative predictive value (NPV) of IMA and CRP for ischemia origin was 79.2% and 38.6%, respectively. IMA may make an early diagnosis of acute coronary ischemia, and will improve the early diagnostic sensitivity and specificity of ACS.

Detection of ciguatoxin in fish tissue using sandwich ELISA and neuroblastoma cell bioassay.

Abstract: The applicability of a new enzyme-linked immunoassay (ELISA) for detecting ciguatoxin (CTX) in fish tissue was evaluated by testing three fish species commonly implicated in ciguatera fish poisoning in Hawaii. A total of 164 individual almaco jack (Seriola rivoliana) and greater amberjack (S. dumerili) and a total of 175 individuals of the blue-spotted grouper (Cephalopholis argus) were caught at various locations in the Hawaiian Islands. Muscle tissue from each individual was assessed for the presence of CTX using two methods: a semi-quantitative ELISA that was recently developed for detecting picogram levels of CTX in fish extract and a neuroblastoma (NB) cell assay commonly used to screen for marine toxins in fish. Results of the tests were highly correlated, with the ELISA indicating the presence of CTX in 9.4% of all fish samples, and the NB assay indicating toxicity in 6.8% of the fish samples. We conclude that the ELISA produces reliable and accurate results that are consistent with those provided by the accepted NB assay and that the ELISA has potential for future applications in screening fish populations for CTX.

Development of an enzyme-linked immunosorbent assay for the detection of ciguatoxin in fish tissue using chicken immunoglobulin Y

Abstract: A sandwich enzyme-linked immunosorbent assay was developed to detect ciguatoxin (CTX) in fish tissue. The assay utilizes two antibodies, chicken immunoglobulin Y specific to the ABCD domain of CTX and a mouse monoclonal immunoglobulin G–hor-seradish peroxidase conjugate specific to the JKLM domain of CTX. The sensitivity, working range, cross reactivity, accuracy, precision, and reproducibility were examined. J. Clin. Lab. Anal. 22:239–245, 2008. © 2008 Wiley-Liss, Inc.

Effects of cryopreservation on CD4+ CD25+ T cells of HIV-1 infected individuals.

Abstract: The role of T regulatory cells (Tregs) in human immunodeficiency virus (HIV)-1 infection, although not entirely clear, has recently been highlighted. Despite their lack of specificity, fluorochrome-labeled CD4 and CD25 antibodies are common flow cytometric reagents used to identify these cells with immunosuppressive potential. Cryopreservation has previously been shown to alter the proportions of lymphocytes with certain phenotypes expressed in peripheral blood mononuclear cells (PBMCs). The aim of this study was to assess the effect of cryopreservation on CD4+ CD25+ T cells in PBMCs from HIV-1+ individuals to guide the design of future studies on Tregs. We recruited 30 HIV-1+ individuals and nine healthy controls. CD25 expression in CD4+ T cells was compared between fresh and frozen/thawed PBMC samples from the same time point. In this study, cryopreservation significantly decreased the proportion of CD4+ CD25+ T cells in PBMC samples from HIV-1 infected subjects. This finding suggests that studies of CD4+ CD25+ T cells should be carried out on fresh samples to avoid bias introduced by cryopreservation.

Effect of hemodialysis on traditional and innovative cardiac markers.

Abstract: The diagnostic approach to acute coronary syndrome (ACS) is challenging in patients with impaired renal function since most serum biomarkers are commonly increased in this clinical setting. Cardiac troponin T (cTnT), creatine kinase isoenzyme MB (CK MB), myoglobin, and ischemia modified albumin (IMA), were assayed in 45 patients prehemodialysis (pre-HD) and posthemodialysis (post-HD), and results were adjusted for hemoconcentration. The pre-HD values of serum IMA and cTnT were above the respective diagnostic thresholds (IMA<85 K units/L; cTnT <0.03 ng/mL) in six (13%) and 27 (60%) patients undergoing chronic HD, respectively. A significant (105.0 vs. 79.0 K units/L, P<0.0001) and variable (+38%; 95% confidence interval [CI], 12-65%) increase of serum IMA was observed post-HD, whereas the other biomarkers significantly decreased (cTnT: 0.029 vs. 0.044 ng/mL, P=0.016; CK-MB: 2.33 vs. 2.50 microg/L, P<0.0001; myoglobin: 128.1 vs. 148.7 microg/L, P<0.0001). Biomarkers of myocardial injury, especially cTnT and IMA, might be used in HD patients, provided that an appropriate diagnostic interpretation is guarantee, according to individual baseine value, metabolism, and time of sampling. Moreover, IMA might be reliably applied to stratify the long-term risk of these patients, but not for diagnosing an ACS during or immediately post-HD.

Protein oxidation markers in the serum and synovial fluid of psoriatic arthritis patients.

Abstract: The role of oxidative stress has been studied in rheumatoid arthritis (RA) and other inflammatory joint diseases to some extent, but its importance in psoriatic arthritis (PsA) has rarely been investigated. The aim of this study was to analyze the levels of protein oxidation markers, sulfhydryl (SH) and carbonyl (CO) groups, in the synovial fluid (SF) and serum of PsA patients and compare them with the findings in RA and osteoarthritis (OA) patients. A total of 49 subjects with a knee-joint effusion including 16 PsA, 18 RA, and 15 OA patients were studied. In all patients, the levels of SH groups measured in the serum and SF inversely correlated with the number of white blood cells (WBC) (P<0.05) and the percentage of polymorphonuclear leukocytes (PMN) (P<0.01) in SF. Serum SH levels inversely correlated with serum erythrocyte sedimentation rate (ESR) (P<0.02) and C-reactive protein (CRP) (P<0.05) values. The SH levels in SF were significantly lower in patients affected by PsA and RA compared to OA cases (P<0.02). The serum SH levels in PsA were lower than OA (P<0.001) and higher than RA patients (P<0.05). The serum and synovial levels of CO groups in PsA, RA, and OA patients were similar. Our study provides novel evidence on the involvement of protein oxidation in PsA and confirms the important role of oxidative stress in the pathogenesis of RA. These data suggest that antioxidant agents can potentially be a useful addition to the conventional therapy in the management of these diseases.

Three‐way comparison of methods for the measurement of the erythrocyte sedimentation rate

Abstract: An erythrocyte sedimentation rate (ESR) is a nonspecific sickness index, which is not diagnostic of any particular disease, but which when elevated may indicate the presence of inflammation, infection, rheumatologic disease, or neoplasm. Technological advances continue to evolve to make this old test to conform to requirements of a modern analytical laboratory. In this evaluation, two new semi-automated ESR measuring systems, the HumaSed and ESR-Auto Plus (EAP), were compared with the Westergren method with regard to the accuracy and precision of ESR measurement, sample stability, and interference of various substances with the ESR assay. Samples from 125 patients were analyzed with the three methods and the results compared using linear regression and Bland-Altman plots. The mean ESR values of the HumaSed (32.10+/-4.86) and EAP (38.09+/-5.33) were comparable to that of the Westergren (31.54+/-4.94). The high correlation coefficients of 0.910-0.96 and the Bland-Altman scatter plots revealed good association and agreement between the three methods. Bias between the three methods was small and the imprecision was within acceptable limits. ESR analysis beyond 4 hr was found to be unacceptable owing to sample instability. There was bilirubin and lipid but not heparin interference in the two automated systems. Overall, two automated analyzers were found to be fast, reliable, standardized, simplified, and safe instruments with accuracy and precision for ESR measurement comparable to the Westergren.

Mass spectroscopic characteristics of low molecular weight proteins extracted from calcium oxalate stones: preliminary study.

Abstract: It is believed that boundary compositions of matrix proteins might play a role in stone formation; however, few proteomic studies concerning matrix proteins in urinary stones have been conducted. In this study, we extracted low molecular weight proteins from calcium oxalate stones and measured their characteristic patterns by mass spectroscopy. A total of 10 stones were surgically removed from patients with urolithiasis. Proteins were extracted from the stones and identified by one-dimensional electrophoresis (sodium dodecyl sulfate buffer [SDS]-polyacrylamide gel electrophoresis [SDS-PAGE]). After in-gel digest, samples were analyzed by the surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) technique. The peptide sequences were analyzed from the data of mass spectroscopy. Proteins were identified from Database Search (SwissProt Protein Database; Swiss Institute of Bioinformatics; http://www.expasy.org/sprot) on a MASCOT server (Matrix Science Ltd.; http://www.matrixscience.com). A total of three bands of proteins (27, 18, and 14 kDa) were identified from SDS-PAGE in each stone sample. A database search (SwissProt) on a MASCOT server revealed that the most frequently seen proteins from band 1 (27 kDa) were leukocyte elastase precursor, cathepsin G precursor, azurocidin precursor, and myeloblastin precursor (EC 3.4.21.76) (leukocyte proteinase 3); band 2 (18 kDa) comprised calgranulin B, eosinophil cationic protein precursor, and lysozyme C precursor; band 3 (14 kDa) showed neutrophil defensin 3 precursor, calgranulin A, calgranulin C, and histone H4. The modifications and deamidations found from the mass pattern of these proteins may provide information for the study of matrix proteins. Various lower molecular weight proteins can be extracted from calcium oxalate stones. The characteristic patterns and their functions of those proteins should be further tested to investigate their roles in stone formation.

p53 codon 72 proline/arginine polymorphism and autoimmune thyroid diseases.

Abstract: p53 protein participates in the processes of apoptosis, which is involved in a number of immunological reactions. In order to test whether the p53 gene could be used as a genetic marker for the prediction of the development of autoimmune thyroid diseases (AITD), we screened, by using polymerase chain reaction (PCR) analysis, for the C (CC_C)/G (CG_C) polymorphism at the p53 codon 72 (Pro 72/Arg 72) to determine the genotypes of 107 Hashimoto's thyroiditis (HT) and 90 Graves' disease (GD) patients, and 105 normal controls. The data demonstrated that, for the genotype analysis, HT patients featured an enhanced numerical ratio for the Arg/Arg homozygous genotype (33.7%) and a diminished ratio for the Arg/Pro heterozygous genotype (41.1%) at the p53 codon 72 than was the case for normal controls (Arg/Arg: 17.1% and Arg/Pro: 61.9%; P=0.005). The odds ratio for the risk of the Arg/Arg genotype's appearance, compared with that of the Arg/Pro and Pro/Pro genotypes combined, for the HT patient group was 2.450 (95% confidence interval: 1.274–4.716). With respect to allelic analysis, we did not observe significant difference in the frequency of appearance of the Arg allelic variant and the Pro allelic variant for the p53 codon 72 when comparing the HT patient group with the control group (P=0.208). On the other hand, GD patients presented no significant difference in distribution for both genotype and allelic frequencies (P=0.344 and 0.245, respectively) when compared with normal controls. In conclusion, HT patients feature a greater ratio of arginine homozygosity at p53 codon 72 than in the case for normal subjects. The p53 codon 72 proline/arginine polymorphism may be a genetic marker to predict the increased susceptibility of development of HT. J. Clin. Lab. Anal. 22:321–326, 2008. © 2008 Wiley-Liss, Inc.

A simple, rapid, and convenient Luminex-compatible method of tissue isolation.

Abstract: Various pathological conditions are associated with changes in multiple protein biomarkers, and these changes can be assessed using xMAP™ beads and the Luminex™ platform. Although this platform is most commonly utilized in the analysis of biomarkers in serum, plasma, or urine, it is often desirable to measure these analytes in tissues (e.g., biopsy specimens). We have developed a simple, rapid method of tissue isolation in which excised tissues are permeabilized in RNAlater™ at 4°C overnight, followed by Luminex™ analysis for inflammatory biomarkers (cytokines). This method was compared with flash-freezing in both mouse liver and human debrided wound specimens, and may be of utility in other clinical settings. J. Clin. Lab. Anal. 22:278–281, 2008. © 2008 Wiley-Liss, Inc.

Comparative evaluation of FASTPlaque assay with PCR and other conventional in vitro diagnostic methods for the early detection of pulmonary tuberculosis

Abstract: Rapid and accurate diagnosis of symptomatic patients of pulmonary tuberculosis (TB) is highly desirable to minimize the spread of the disease in the society. We, therefore, compared the usefulness of various conventional diagnostic methods, the in-house polymerase chain reaction (PCR), and the FASTPlaque assay in this study. Laboratory data of 150 patients with clinical diagnosis of pulmonary TB and 50 controls were included in this study. The sputa from all these 200 individuals were subjected to acid-fast staining, culture on Lowenstein-Jensen (L-J) slants, automated BACTEC-MGIT-960 culture methods, and a mycobacteriophage assay. A mycobacterium genus and Mycobacterium tuberculosis species-specific PCRs were also done and samples positive on both PCRs were considered as standard for comparison. Of the 5 in vitro diagnostic tests, PCR method was found to be the most rapid, sensitive, and specific, detecting all the 150 cases of pulmonary TB without any false-positive and negative result. In comparison with PCR the sensitivity of MGIT-960 was 90%, followed by FASTPlaque assay (76.7%), L-J culture method (73.3%), and microscopy (60%). The mean detection time for smear-positive and smear-negative samples was 12.5 and 14 days in MGIT-960 and 18 and 25 days for L-J method, respectively. The FASTPlaque failed to detect mycobacteria from the paucibacillary samples. The contamination rates for MGIT-960, L-J, and FASTPlaque assays were 4, 8 and 10%, respectively. The best correlation with mycobacterial load in the specimen was observed in BACTEC-MGIT-960 showing 66.6% detection rate in paucibacillary, 83.3% in 1+ samples, and 100% in 2+ and 3+ samples. Out of the 150 patients, 140 (93.3%) could be diagnosed by one or more nonmolecular methods. Therefore, it could be concluded that combination of three or more in vitro diagnostic methods will have acceptable detection level.

Detection and significance of serum protein markers of small-cell lung cancer.

Abstract: Currently, no satisfactory biomarkers are available to screen for small-cell lung cancer (SCLC). We applied a surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) ProteinChip system to detect 150 serum samples (including 54 SCLC patients, 24 non-small cell lung cancer [NSCLC] patients, 32 pneumonia patients, and 40 healthy individuals). The spectra data were analyzed by support vector machine (SVM) and potential biomarkers were chosen for the system training and used to construct diagnostic model. Pattern 1, constructed of four protein peaks with mass/charge (m/z) of 4,293 Da, 4,612 Da, 6,455 Da, and 7,582 Da, separated SCLC patients from the healthy individuals with a sensitivity of 88.9% and a specificity of 85.7%. This pattern performed significantly better than the current marker, neuron-specific enolase (NSE) (P<0.05). Pattern 2, constructed of protein peaks with mass/charge (m/z) of 2,764 Da and 1,7368 Da, separated SCLC from pneumonia with a sensitivity of 88.9% and a specificity of 91.7%. Pattern 3, constructed of another three protein peaks with m/z of 3,912 Da, 7,562 Da, and 13,777 Da, separated SCLC from NSCLC. The sensitivity and specificity were 83.3% and 75.0%, respectively. These results suggested that SELDI-TOF MS combined with support vector machine yields significantly higher sensitivity and specificity for the detection of serum protein of SCLC.

Preliminary clinical measurement of the expression of TNF‐related apoptosis inducing ligand in patients with ankylosing spondylitis

Abstract: It has recently been reported that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) plays various roles in such autoimmune diseases as diabetes, multiple sclerosis (MS), and systemic lupus erythematosus (SLE). However, it has still remained unclear whether there is a close relationship between TRAIL and ankylosing spondylitis (AS). In this study, we investigated the association between the expression of TRAIL and AS. The specific messenger ribonucleic acid (mRNA) levels of TRAIL in peripheral blood mononuclear cells (PBMCs), serum sTRAIL, and TNF-α concentrations from 60 AS patients, 20 rheumatoid arthritis (RA) patients, and 30 healthy controls were determined by real-time fluorescent quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The results indicated that the expression levels of TRAIL mRNA, and serum sTRAIL were significantly elevated in AS patients, compared with RA patients and healthy controls, and there was a close association between TRAIL mRNA and sTRAIL levels. However, there was no significant difference between human leukocyte antigen (HLA)-B27-positive and -negative AS patients. In HLA-B27-positive patients, TRAIL mRNA and sTRAIL closely correlated with serum TNF-α and C-reactive protein (CRP), but did not correlate in HLA-B27-negative patients. In conclusion, upregulated expression of TRAIL might be somewhat specific for evaluation of AS. J. Clin. Lab. Anal. 22:138–145, 2008. © 2008 Wiley-Liss, Inc.

SNP analysis using CataCleave probes.

Abstract: CataCleave probes are catalytically cleavable fluorescence probes having a chimeric deoxyribonucleic acid (DNA)-ribonucleic acid (RNA)-DNA structure that can be used for real-time detection of single nucleotide polymorphisms (SNPs), insertions, and deletions. Fluorescent donor emission is normally quenched by Forster resonance energy transfer (FRET). Upon binding to the target, if the RNA/DNA hybrid is correctly base-paired, ribonuclease (RNase) H will cleave the RNA moiety and the probe fragments will dissociate. FRET is lost and the donor fluorescence signal is recovered. A single-base mismatch within the hybrid region causes probe cleavage to be significantly reduced. We designed CataCleave probes to detect SNPs located in the insulin-like growth factor 2 (IGF-2) gene and at position 702 within the NOD2/CARD15 gene. Probes were also designed to detect a six-basepair deletion in the amelogenin gene and a partially methylated target DNA. Discrimination between wild-type and SNP is demonstrated for both genes in homogeneous reactions under isothermal and temperature cycling conditions. These probes were also able to identify a multibase deletion and methylated DNA. Cleavage rates were proportional to target concentration. Probe length and position of fluorescent labels may also be modified for use in multiplexing high-throughput SNP assays. This represents a novel method for the detection of SNPs.

Liver function tests in patients with bacteremia

Abstract: The purpose of this study was to evaluate liver function tests as potential indicators of bacteremia. We examined 156 patients with laboratory-confirmed bacteremia (bacteremia group) and 211 bacteremia-negative patients with bacterial infections (control group). The patients of the two groups had no underlying liver diseases. For patients in the bacteremia group, we analyzed liver function tests results obtained the day when the first positive blood culture was ordered. For those in the control group, the same data were obtained on the day when the first of multiple negative blood cultures was ordered. At t-test analyses, serum levels of gamma-glutamyl transpeptidase (gamma-GT) and alkaline phosphatase (ALP) were significantly higher, and those of albumin, total cholesterol, and cholinesterase were significantly lower in the bacteremia group than in the control group. Multivariate analyses found serum cholinesterase as an independent factor with adjusted odds ratio of 0.319 (per 65 U/L, standard deviation [SD] size). Serum level of C-reactive protein (CRP), on the other hand, showed no significant difference between the two groups. Serum levels of gamma-GT, ALP, albumin, total cholesterol, and cholinesterase more rapidly altered when various bacterial infections accompanied bacteremia. Therefore, they may be useful in detecting sepsis in its early stages.

Development of multiple complications in type 2 diabetes is associated with the increase of multiple markers of chronic inflammation.

Abstract: Patients with type 2 diabetes (T2DM) are known at risk for developing cardiovascular disease (CVD), nephropathy, and cancer. We were interested to find out whether multiple markers associated with chronic inflammation are detectable in patients with T2DM and are increased in patients with T2DM who developed additional clinical complications. A sequence of multiple risk markers for atherogenesis, associated with chronic inflammation, was measured in patients with T2DM before and after the development of clinical complications. We found that multiple clinical complications frequently developed simultaneously in patients with T2DM. At the early stage of T2DM, only low levels and low percent elevations of multiple risk markers were detected. However, both the level and the percent elevation of these markers were found to increase with disease progression and the development of clinical complications. We believe that chronic inflammation not only contributes to the pathogenesis of T2DM but also continues to increase in T2DM patients who are developing additional clinical complications. It appears that these multiple markers are potentially useful not only for monitoring the progression of T2DM but also predicting the risk of developing macro- and microvascular disease, nephropathy, and cancer. J. Clin. Lab. Anal. 22:6–13, 2008. © 2008 Wiley-Liss, Inc.