Microparticle Enzyme Immunoassay Method Research Articles

Tacrolimus trough level and oxidative stress in Tunisian kidney transplanted patients

Background The effect of tacrolimus (TAC) on oxidative stress after kidney transplantation (KT) is unclear. This study aimed to evaluate the influence of TAC trough levels of oxidative stress status in Tunisian KT patients during the post-transplantation period (PTP). Methods A prospective study including 90 KT patients was performed. TAC whole-blood concentrations were measured by the microparticle enzyme immunoassay method and adjusted according to the target range. Plasma levels of oxidants (malondialdehyde (MDA) and advanced oxidation protein products (AOPP)) and antioxidants (ascorbic acid, glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD)) were measured using spectrophotometry. The subjects were subdivided according to PTP into three groups: patients with early, intermediate, and late PT. According to the TAC level, they were subdivided into LL-TAC, NL-TAC, and HL-TAC groups. Results A decrease in MDA levels, SOD activity, and an increase in GSH levels and GPx activity were observed in patients with late PT compared to those with early and intermediate PT (p < 0.05). Patients with LL-TAC had lower MDA levels and higher GSH levels and GPx activity compared with the NL-TAC and HL-TAC groups (p < 0.05). Conclusion Our results have shown that in KT patients, despite the recovery of kidney function, the TAC reduced but did not normalize oxidative stress levels in long-term therapy, and the TAC effect significantly depends on the concentration used.

EFEK FORTIFIKASI VITAMIN B12 TERHADAP KADAR VITAMIN B12 SERUM DAN HOMOSISTEIN SERUM PADA VEGETARIAN

Introduction: Vegetarians consume plant-based foods with or without eggs and milk. Vegetarians are at risk of vitamin B12 deficiency, as natural sources of vitamin B12 are limited to animal-based foods. Vitamin B12 deficiency can lead to megaloblastic anemia, nerve damage and increase homocysteine level. Higher homocysteine level can increase the risk of coronary heart disease and stroke. The objective of this study was to investigate the effect of vitamin B12 fortification on the level of serum vitamin B12 and homocysteine in vegetarian. Method: The research design was an experimental study, community trial. The samples were 42 vegetarians with vitamin B12 deficiency (&lt; 156 pmol/L) selected from 118 vegetarians as members of Indonesia Vegetarian Society (IVS) Pekanbaru, treated by vitamin B12 fortified oatmeal for three months from March to June 2010. Serum vitamin B12 and homocysteine were measured by electrochemiluminescent immunoassay and microparticle enzyme immunoassay method respectively.&#x0D; Result: Prevalence of vitamin B12 deficiency in vegetarian was 35.6%. Statistical analysis showed a significant increase of serum vitamin B12 from 124.6 to 284.6 pmol/L (p=0.001) and significant decrease of serum homocysteine from 20.1 to 15.1 µmol/L (p=0.001).&#x0D; Conclusion: Consumption of vitamin B12 fortified oatmeal increases the level of serum vitamin B12 and decreases the level of serum homocysteine significantly in vegetarian with vitamin B12 deficiency.

CYP3A5 polymorphisms and their effects on tacrolimus exposure in an ethnically diverse South African renal transplant population.

Tacrolimus forms the cornerstone for immunosuppression in solid-organ transplantation. It has a narrow therapeutic window with wide inter- and intra-patient variability (IPV). Cytochrome P-450 3A5 (CYP3A5) is the main enzyme involved in tacrolimus metabolism, and rs776746A&gt;G is the most frequently studied polymorphism in the CYP3A5 gene. The rs776746A&gt;G (i.e. CYP3A5*3) single-nucleotide polymorphism in CYP3A5 alters tacrolimus predose trough concentration (C0) and may also affect IPV, which may lead to immune- and/or drug-mediated allograft injury. CYP3A5*3 may result in absent (*3/*3), partial (*1/*3) or normal (*1/*1) CYP3A5 expression. The effect of CYP3A5*3 on tacrolimus exposure and variability has not been examined in South African (SA) transplant recipients. To determine the frequencies and effect of CYP3A5 and adenosine triphosphate-binding cassette subfamily B member 1 (ABCB1) polymorphisms on tacrolimus C0/dose ratios in different ethnic groups attending a tertiary renal transplant clinic in SA, and other factors that may explain inter- and IPV in tacrolimus C0. All consenting stable renal transplant recipients on tacrolimus at the Livingstone Hospital Renal Unit in Port Elizabeth, SA, were included. Tacrolimus concentrations were obtained using a microparticle enzyme immunoassay method (ARCHITECT analyser, Abbott Laboratories). Polymerase chain reaction/restriction fragment length polymorphism was used to genotype for CYP3A5*3 and *6 allelic variants. There were 43 participants (35% black African, 44% mixed ancestry and 21% white), with a mean age of 44.5 years, median duration post-transplant of 47 months and median (interquartile range) creatinine and estimated glomerular filtration rate levels of 118(92- 140) µmol/L and 62 (49 - 76) mL/min at study inclusion. The mean tacrolimus C0 in the study was 6.7 ng/mL, with no difference across the different ethnic groups. However, the mean total daily dose of tacrolimus required was 9.1 mg (0.12 mg/kg), 7.2 mg (0.09 mg/kg) and 4.3 mg (0.06 mg/kg) in black, mixed-ancestry and white patients, respectively (p=0.017). The frequencies for CYP3A5 expressors (i.e. CYP3A5*1/*1 + CYP3A5*1/*3 genotypes) were 72%, 100%, 76% and 12% for all patients combined and black, mixed-ancestry and white patients, respectively. The frequencies for CYP3A5 non-expressors (i.e. CYP3A5*3/*3 genotypes) were 0%, 24% and 88% among the black, mixed-ancestry and white patients, respectively. None of the patients carried the CYP3A5*6 allele. CYP3A5*1/*1 and CYP3A5*1/*3 genotype carriers required a two-fold increase in dose compared with the non-expressor genotype carriers, CYP3A5*3/*3 (p&lt;0.05). CYP3A5*3/*3 carriers also demonstrated higher IPV than CYP3A5*1/*1 and *1/*3 carriers (18.1% v. 14.2%; p=0.125). Compared with global transplant populations, SA renal transplant recipients demonstrated a very high rate of CYP3A5 expression, with a significant impact on tacrolimus pharmacokinetics. Genetic variation in CYP3A5 expression affects tacrolimus dosing requirements, and knowing the CYP3A5 genotype of transplant patients may allow better dose prediction compared with current standard dosing recommendations in a multi-ethnic population. Overall, black African patients required higher doses of tacrolimus than their white counterparts. While further prospective studies are needed to better evaluate dosing algorithms, it would appear that the starting dose of tacrolimus should be higher in black and mixed-race patients.

Gender-dependent predictable pharmacokinetic method for tacrolimus exposure monitoring in kidney transplant patients

Tacrolimus (Tac) is an immunosuppressive drug with a narrow therapeutic width and highly variable pharmacokinetics. Therefore, monitoring of Tac blood concentrations is of utmost importance in the management of renal transplant recipients. The occurrence and intensity of adverse effects depend on blood concentration and total exposure of the organism to this drug. This implies finding a new gender-dependent predictable method for Tac exposure monitoring based on determination of the area under the time concentration curve (AUC). The primary aim of this study was to investigate gender differences in systemic body exposure to Tac in renal transplant patients after the first oral dose and in a steady state by determining 12-h AUC (AUC(0-12)). The secondary objective was to find the best sampling time in which measured Tac concentration best predicts AUC value with respect to gender. Tac pharmacokinetic study was conducted in 20 kidney transplant recipients (10 men/10 women) on quaternary immunosuppressive therapy. The first oral Tac dose (0.05 mg/kg) was given on the fifth day post-transplant. After reaching steady state, regimen stabilized and dosage was adjusted in accordance with the level of Tac. Blood concentrations were measured by microparticle enzyme immunoassay method. AUC(0-12) for each patient was calculated after the first oral Tac dose and in the steady state from a plot of Tac concentration versus time from 0 to 12 h using the trapezoid rule. Associations between each sampling time point of concentrations within 12 h after the administration and AUC(0-12) were evaluated by Pearson correlation coefficients. Abbreviated sampling equations were derived by multiple stepwise regression analyses. Statistically significant difference was found in AUC(0-12) between male and female patients after the first oral dose (p < 0.01), but this difference was lost in a steady state. In female recipients C(2) seemed to be good indicator of total body exposure to Tac after the first oral dose and this was also confirmed in a steady state. The three-point sampling method was required for calculating AUC after the first oral dose in male patients, whereas in the steady state, concentration of C(8) seemed to be a good indicator of abbreviated AUC for a Tac monitoring strategy in male patients. Non-compartment Tac pharmacokinetic and regression analysis showed gender difference in total Tac exposure and determined the best predictable Tac concentrations after the first oral dose. Our study confirmed gender-dependent pharmacokinetics in a steady state in terms of best sampling time in which measured Tac concentration best predicts AUC value.

HBsAg, anti-HCV, anti-HIV 1/2 and syphilis seroprevalence in healthy volunteer blood donors in southeastern Anatolia

This study investigated the seroprevalence of hepatitis B virus surface antigen (HBsAg), antibody against hepatitis C virus (anti-HCV), antibody against human immunodeficiency virus type 1/2 (anti-HIV 1/2), and antibody against Treponema pallidum (anti-Treponemal or syphilis antibody) in healthy volunteer blood donors, and assessed their distribution according to the years and genders. HBsAg, anti-HCV, anti-HIV ½, and syphilis screening results of a total of 266,035 healthy volunteer blood donors who had been admitted for blood donation to the Regional Blood Center of Dicle University Hospital between January 2000 and December 2010 were evaluated, retrospectively. HBsAg, anti-HCV, and anti-HIV 1/2 screening were performed using a fully automated device with the microparticle enzyme immunoassay method (MEIA). Syphilis screening was performed by Rapid Plasma Reagin (RPR) carbon test between January 2000 and December 2009, and by using a fully automated device with the MEIA method between January 2010 and December 2010. Of 266,035 healthy volunteer blood donors, 259,384 (97.5%) were male and 6,651 (2.5%) were female. Statistically, there was not any significant difference between male and female genders for HBsAg, anti-HCV and syphilis seropositivities (P = 0.729, P = 0.748, and P = 0.861, respectively). HBsAg was found to be positive in 8,422 (3.17%), anti-HCV in 1,703 (0.64%), anti-HIV 1/2 in one (0.0004%) of 266,035 healthy volunteer blood donors, and syphilis antibody with RPR in 166 (0.07%) of 246,341 healthy volunteer blood donors. Blood donor forms should be carefully tailored to improve the identification of possible risks of transfusion-transmitted infections.

Anthropometric profile of HIV-uninfected and HIV-infected women aged 25–44 years in Mangaung, Free State

Background: Obesity and human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) affect significant numbers of black women in South Africa.Method: Using township maps, a random sample of 500 black women residing in Mangaung in the Free State was selected to participate in this study in the year 2000. The women were divided into two age groups, namely 25–34 years (n = 273) and 35–44 years (n = 215). Anthropometric measurements, including height, weight [to calculate the body mass index (BMI)] and waist circumference (WC) were taken. Fat percentage was measured with bioelectrical impedance. HIV status was determined using a microparticle enzyme immunoassay method. Socio-demographic status, health status, dietary intake, level of physical activity, body perception and attitude toward weight control, as well as prevalence and risk of lifestyle diseases were determined as part of the larger study.Results: Sixty-one per cent of younger women (25–34 years) and 38% of older women (35–44 years) were infected with HIV. In younger HIV-infected women, median BMI, WC and fat percentage were significantly lower than in HIV-uninfected women.Conclusions: HIV infection rates were found to be higher among younger than older women. The prevalence of obesity was high overall. Median BMI values ranged between 24.4 kg/m2 and 27.6 kg/m2. A large percentage of all women fell in the unhealthy fat percentage category (excessive body fat), ranging between 65.9% of HIV-infected young women and 79.3% of older HIV-infected women. HIV, even in the asymptomatic stage, influences anthropometric indicators.

Gender Differences in Pharmacokinetics of Tacrolimus and Their Clinical Significance in Kidney Transplant Recipients

The possible influence of gender on tacrolimus disposition and response in kidney transplant recipients is an issue of medical importance. The aim of this study was to detect interpatient pharmacokinetic variability of tacrolimus due to patients' gender and to assess the predictability of individual tacrolimus concentrations using abbreviated AUC measurements. The secondary objective was to find the best sampling time to predict the exposure of tacrolimus in kidney transplant recipients. Gender-related first oral dose tacrolimus pharmacokinetics studies were conducted in 20 Serbian kidney transplant recipients (10 men/10 women) on quaternary immunosuppressive therapy. The first tacrolimus oral dose (0.05 mg/kg) was given on day 5 post-transplant. Blood concentrations were measured by microparticle enzyme immunoassay method. Associations between each sampling time point of concentrations and 12 hours after the administration AUC (AUC(0-12)) were evaluated by Pearson correlation coefficients. Abbreviated sampling equations were derived by multiple stepwise regression analyses. AUC(0-12) showed remarkable interindividual variations after the first tacrolimus oral dose. There were significantly lower values of AUC in women than men (P < 0.05). The most important time point influencing AUC(0-12) was the concentration of tacrolimus measured 2 hours after administration(C(2)) in women, whereas in men the most important time points were the concentrations at 1 (C(1)), 4 (C(4)), and 12 (C(12)) hours as an abbreviated AUC. Our results show significant differences between men and women. C(2) seems to be indicator of total body exposure to tacrolimus in the early period after kidney transplant in women. The three-point sampling method seems to be a good indicator of abbreviated AUC for a tacrolimus monitoring strategy in men.

Clinical Pharmacokinetics of Tacrolimus after the First Oral Administration in Renal Transplant Recipients on Triple Immunosuppressive Therapy

Monitoring of tacrolimus blood concentration is of utmost importance in the management of renal transplant recipients because of Narrow Therapeutic Index and highly variable pharmacokinetics. The aim of this study was to detect inter-patient pharmacokinetic variability of tacrolimus and to assess the predictability of individual tacrolimus concentrations at various times of the area under the curve (AUC) seeking to find the best sampling time to predict the exposure of tacrolimus in renal transplant recipients with triple therapy. This oral dose tacrolimus pharmacokinetics study was conducted in 18 Serbian renal transplant recipients on triple immunosuppressive therapy, including basiliximab. The first oral dose of tacrolimus (0.05 mg/kg) was given on day 5 post-transplant; blood concentration was measured by microparticle enzyme immunoassay method. Associations between each sampling time-point of concentrations and AUC(12) were evaluated by Pearson correlation coefficients. Abbreviated sampling equations were derived by multiple, stepwise regression analyses. The variance in the strength of association between predicted AUC (AUC(p)) and AUC(12) was reflected by linear regression coefficients. AUC(12) showed remarkable inter-individual variations after the first oral dose of tacrolimus. The area of the maximum AUC was four times higher than that of the minimum AUC. C(4) seems to be an indicator of total body exposure to tacrolimus. Alternatively, the concentrations at 1.5, 4 and 8 hr as an abbreviated AUC were as good a predictor as a full pharmacokinetic study. Our results show a significant difference between men and women. A three-point sampling method seemed to be the best abbreviated AUC for a cost-effective tacrolimus monitoring strategy.

Role of serum troponin-I in identifying left ventricular ejection fraction of

To determine the serum levels of troponin-I in identifying left ventricular ejection fraction (LVEF) of <or= 40% in patients with first anterior ST Elevation Myocardial Infarction (STEMI). Case series. At the Tabba Heart Institute, Karachi, from May to November 2008. Consecutive patients presenting with first anterior STEMI were studied. Troponin-I concentration was measured by MEIA (microparticle enzyme immunoassay) method and LVEF was visually assessed. Analysis of relation between troponin-I levels and LVEF by the Receiver-Operator Characteristic (ROC) curve was performed to determine the cut off values of troponin-I in identifying LVEF of <or= 40% in patients, who had received streptokinase or undergone primary Percutaneous Coronary Intervention (PCI). Out of the 90 patients studied, 50 patients received streptokinase and the remaining 40 patients underwent primary PCI. Mean age was 54.6 +/- 9 years and 82% were male. Troponin-I levels of > 63.5 ng/ml predicted LVEF of <or= 40% with a sensitivity of 94% and specificity of 97% in patients receiving streptokinase, whereas in patients undergoing primary PCI, troponin-I levels of > 87.5 ng/ml predicted LVEF < 40% with a sensitivity of 86% and specificity of 100%. Troponin-I concentration of > 63.5 ng/ml and > 87.5 ng/ml can predict LVEF <or= 40% in patients treated with either streptokinase or primary PCI respectively for first anterior STEMI. Troponin-I can be used as a non-imaging tool to identify patients with LVEF <or= 40% who need early aggressive therapy.

Is the Affinity Column–Mediated Immunoassay Method Suitable as an Alternative to the Microparticle Enzyme Immunoassay Method as a Blood Tacrolimus Assay?

Background Tacrolimus is a potent immunosuppressive drug used in organ transplantation. Because of its substantial toxic effects, narrow therapeutic index, and interindividual pharmacokinetic variability, therapeutic drug monitoring of whole-blood tacrolimus concentrations has been recommended. We investigated the comparability of the results of 2 immunoassay systems, affinity column–mediated immunoassay (ACMIA) and microparticle enzyme immunoassay (MEIA), comparing differences in the tacrolimus concentrations measured by the 2 methods in relation to the hematologic and biochemical values of hepatic and renal functions. Methods A total of 154 samples from kidney or liver transplant recipients were subjected to Dimension RxL HM with a tacrolimus Flex reagent cartilage for the ACMIA method and IMx tacrolimus II for the MEIA method. Results Tacrolimus concentrations measured by the ACMIA method (n = 154) closely correlated with those measured by the MEIA method ( r = 0.84). The Bland-Altman plot using concentration differences between the 2 methods and the average of the 2 methods showed no specific trends. The tacrolimus levels determined by both the MEIA method and the ACMIA method were not influenced by hematocrit levels, but the difference between the 2 methods (ACMIA − MEIA) tended to be larger in low hematocrit samples ( P < .001). Conclusion The ACMIA method used for a tacrolimus assay is precise and has advantages, including the lack of a required pretreatment procedure. Furthermore, it is only slightly influenced by the hematologic or biochemical status of the samples.

Micafungin Does Not Influence the Concentration of Tacrolimus in Patients after Allogeneic Hematopoietic Stem Cell Transplantation.

Purpose: Tacrolimus (Tac) is commonly used for stem cell transplant (SCT) recipients as an agent for prophylaxis of graft-versus-host disease (GVHD) and because of its narrow therapeutic index a close drug monitoring is needed. Micafungin (MCFG) is widely used as a strong antifungal agent in empirical therapy for patients with febrile neutropenia. Both Tac and MCFG are substrates of CYP3A4 in vitro. Therefore, there is a risk of drug interaction by the coadministration of these 2 agents.Methods: Five consecutive Japanese patients (3 males and 2 females) with hematological malignancies who received allogeneic SCT in our hospital between August 2005 and December 2006 were enrolled in this study. We estimated the drug interaction of Tac and MCFG by evaluating the pharmacokinetics in 5 cases of allogeneic SCT. The patients initially received 24 hour continuous intravenous administration of Tac at 0.01 mg/kg. The dose was then adjusted to a concentration between 10–12 ng/ml by monitoring of the concentration 3 times a week. MCFG was started at 150 mg/body/day as an empirical therapy in order to manage the neutropenic fever suggesting fungal infection. MCFG was administered as a one hour infusion. For the determination of both Tac and MCFG concentration, serial blood samples were drawn from the patients at 1, 4, 8 and 24 hours after the initiation of therapy. Tac concentrations were determined by the microparticle enzyme immunoassay method using an IMx Tacrolimus II analyzer (Abbott Diagnostics, Abbott Park, IL). The concentration of MCFG in blood was analyzed by high performance liquid chromatography (HPLC). HPLC was performed using a Phenomenex Luna 3μ C18 column (4.6 mm x 150 mm) as an analytical column.Results: Five patients were included in this study; 3 were male and 2 were female. The age of patients ranged from 37 to 71 years, with a median age of 53 years. The donor source was unrelated bone marrow (BM) in 1 patient, peripheral blood stem cells (PBSC) from a related donor in 1 patient, and cord blood (CB) in 3 patients. The mean value of the concentration/dose (CD) ratio of Tac in all patients administered concomitant MCFG was 704.7 ± 61.8 ((ng/mL)/(mg/(kg x day)) and the value without MCFG was 710.3 ± 390.4 ((ng/mL)/(mg/(kg x day)). The proportion of the CD ratio in patients treated with Tac and MCFG to the CD ratio in patients treated with Tac alone was 0.886, 0.960, 1.08 and 1.04 for 1, 4, 8 and 24 hours, respectively. For both Tac and MCFG, the 90% confidence intervals for the primary PK parameters (i.e., the CD ratio at each time point) ranged from 80% to 125%. The concentrations of MCFG at 1, 4, 8 and 24 hours after the start of infusion were 27.3, 20.0, 16.7 and 9.4, respectively. Pharmacokinetic parameters of MCFG was following; AUC (area under the curve) 368 ± 172 mg x hr/L, Vd (distribution volume in elimination phase) 0.223 ± 0.106 L/kg, clearance 0.0088 ± 0.003 L/(hr x kg) and elimination half life 18.3 ± 1.7 hr.Conclusion: We concluded that there was no drug interaction between Tac and concomitantly administered MCFG in SCT recipients.

Helicobacter felis Infection Causes an Acute Iron Deficiency in Nonpregnant and Pregnant Mice

The role of Helicobacter pylori infection in iron deficiency during pregnancy is limited. The aim of the present study was to assess the relationship between Helicobacter infection and levels of iron stores in pregnant mice. Female C57BL/6 mice were either inoculated with 10(8) H. pylori, Helicobacter felis or water. In the nonpregnant study, 15 mice from each group were sacrificed after 4 and 20 weeks of infection. In the pregnancy study, after 6 weeks of infection all female mice were mated and approximately 2 weeks after mating, half of the pregnant mice (n = 9/group) from each group were sacrificed. The remaining mice were allowed to give birth, and approximately 4 weeks after birth, mice were asphyxiated with CO2, followed by heart puncture, and killed by cervical dislocation. Serum ferritin and iron were determined with a micro-particle enzyme immunoassay method and by a timed-endpoint method. Serum iron levels in mice infected with H. felis were significantly (p < .05) lowered compared to control (24%) and H. pylori (27%)-infected mice at 4 weeks of infection. Serum iron in the control, H. pylori and H. felis groups were significantly (p < .05) elevated at 20 weeks by 39, 26 and 77%, respectively, compared to 4 weeks of infection. H. felis-infected mice had a significantly (p < .05) decreased serum ferritin level during pregnancy (61%) compared to H. pylori-infected mice. These results suggest that H. felis but not H. pylori infection causes an acute iron deficiency in normal and pregnant mice.

Switching from HPLC/UV to MEIA for whole blood sirolimus quantitation: comparison of methods

Sirolimus is a immunosuppressive agent for renal transplant recipients. Monitoring of whole blood sirolimus concentration is necessary in order to improve clinical outcomes. An increasing number of clinical laboratories (4-14% during 2005) are using microparticle enzyme immunoassay (MEIA) for sirolimus quantitation but previous reports indicated a high variability, with a mean difference of 17% for MEIA method vs. high-performance liquid chromatography/ultraviolet (HPLC/UV). This study was aimed at comparing the reliability of MEIA with the HPLC/UV method. Blood samples from transplant patients were processed using both HPLC/UV and MEIA assays. Comparison and Bland-Altman plots, as well as regression analysis and paired t-test were used to compare results of the assays. Concentrations were stratified into three groups and used to investigate whether any observed difference between methods could be influenced by sirolimus concentration. Regression analysis yielded a coefficient of correlation R of 0.9756, the line of best fit being y=0.9832x+0.1976. The statistical analysis showed no difference between the two sets of experimental data. The average percentage difference between the two methods was found to be -0.2+/-19.2%. On the basis of our present results, the tested MEIA assay is able to quantify sirolimus concentration with a clinically acceptable imprecision, similar to that of HPLC/UV method.

Evaluation of a New Immunoassay to Measure Sirolimus Blood Concentrations Compared to a Tandem Mass-Spectrometric Chromatographic Analysis

A new immunoassay of sirolimus based on the microparticle enzyme immunoassay (MEIA) principle has been developed with collaboration of Abbott Diagnostics. Laboratories and Axis-Shield. Our laboratory evaluated this new assay on 153 whole blood samples (EDTA) drawn from a population of renal ( n = 141) and hepatic ( n = 12) transplant patients. Each blood sample was analyzed simultaneously by MEIA (Y) and by a reference method (X) used routinely in our laboratory, namely, liquid chromatography tandem mass spectrometry (LC-MS/MS). The statistical analysis of Passing-Bablok produced the following results: Spearman r value = 0.95, slope = 1.15 and intercept with the Y axis = +0.2 ng/mL. The observed global overestimation of 15% compared to the reference method could be explained by the cross-reactivity of sirolimus metabolites with the antibody. A complementary analysis taking into account the transplanted organ (kidney versus hepatic) did not show any significant difference between the populations, most likely owing to the low number of hepatic transplantation samples. The analytical performance of the MEIA method showed CV ≤10% and a limit of quantification of 1.5 ng/mL, which were acceptable for routine clinical monitoring. In conclusion, the MEIA method has shown robust, stable, reproducible, features with an excellent correlation with the reference LC-MS/MS method.

Hepatitis C virus infection in psoriatic arthritis

To evaluate the prevalence of hepatitis C virus (HCV) infection in patients with psoriatic arthritis (PsA), compared with patients affected by non HCV-related rheumatic degenerative disorders. One-hundred consecutive subjects with PsA, and a statistically comparable group of 100 consecutive patients with peripheral osteoarthritis (OA) or sciatica due to L4-L5 or L5-S1 herniated disc were tested for HCV infection with a third-generation microparticle enzyme immunoassay (MEIA). Positive cases were submitted to a third-generation recombinant immunoblot assay (RIBA) confirmatory test. Comparison between the HCV prevalence obtained in the 2 enrolled groups was performed using Fisher's exact test. Anti-HCV antibodies were found with the MEIA method, in 1 patient with PsA, and in 4 patients with OA or sciatica. The RIBA method confirmed MEIA results in all positive patients. The difference in HCV prevalence detected in the PsA group and in the control group was not statistically significant (P = 0.68). Furthermore, HCV prevalence in PsA patients was lower than the ones reported in different geographic areas of Italy. Our present report does not confirm previous data that indicated an increased prevalence of HCV in PsA patients, and as a consequence, does not sustain a possible trigger role of HCV in cases of PsA. The absence of clinical or instrumental resources that consent a definite differential diagnosis between PsA and HCV-related arthritis was outlined and analyzed.

Protein interference in thyroid assays: an in vitro study with in vivo consequences

Background Pathological concentration of plasma proteins may cause problems in immunoanalytics. The low triiodotyronine (T3) and thyroxine (T4) levels, frequently found in seriously ill patients, may be ascribed either to laboratory artifact due to the lower thyroid hormone binding capacity or to a compensatory response of the organism to the disease. Methods The authors performed an in vitro experiment, in which sera of seriously ill patients with either low immunoglobulin G (IgG), and/or low albumin levels were investigated for free thyroid hormones (fT3, fT4) following stepwise adjustment of the serum IgG and/or albumin. All two hormones were measured with two different automated immunoassays: the microparticle enzyme immunoassay (MEIA) with two steps (AxSym, Abbott, USA) and the electrochemiluminescence immunoassay (ECLIA). Results The bias of fT3 and fT4 exhibited positive correlations with serum IgG and albumin. The bias of fT3 was more pronounced than that of fT4 following the addition of albumin (40–150% and 10–40%, respectively) as well as following the addition of IgG (8–30% and 0–8%, respectively). The MEIA method was more sensitively affected in case of fT4, whereas the bias of fT3 was more influenced in the ECLIA assay. In MEIA assay, the influence of albumin on the bias of fT3 and fT4 was stronger if serum IgG levels were low. Conclusion The results confirm that pathological thyroid findings in seriously ill patients may largely be ascribed to some laboratory artifacts.

Prevalência de soropositividade para toxoplasmose em gestantes

To measure the prevalence of seropositivity for toxoplasmosis in pregnant women and evaluate its association with maternity age, skin color, place of residence and education. A cross-sectional study in 1,261 pregnant women cared for at the maternity ward of Hospital Nossa Senhora da Conceição was performed from June to October and in December 2000. Serological tests for toxoplasmosis were performed during their pregnancy or delivery. The variables considered were place of residence, skin color, education and serum tests for toxoplasmosis (IgG and IgM). Microparticle Enzyme Immunoassay method (MEIA) was used. The prevalence of seropositivity for toxoplasmosis in the pregnant women studied was of 59.8% (95% CI: 57.0% - 62.5%). An increase in seropositivity in relation to the mother's age was observed (p = 0.012). On the other hand, a higher educational level was found to be a protective factor against toxoplasmosis (p < 0.001). The hypothesis that the proportion of pregnant seropositive women would increase the farther they lived from capital cities was not confirmed (p = 0.750). Differences regarding race were not observed (p = 0.228). In the multivariate analysis, maternity age presented a linear association with the increase of seropositivity, even after adjustment for education, place of residence and skin color. The prevalence of seropositivity in the pregnant women studied is high and justifies the adoption of some primary and secondary preventive measures, until subsequent studies provide greater evidence concerning the rationalization of the diagnostic and therapeutic techniques regarding toxoplasmosis in pregnant women.

Persistence of spermatozoa and prostatic acid phosphatase in specimens from deceased individuals during varied postmortem intervals.

The survival of spermatozoa and the persistence of prostatic acid phosphatase has been an area of interest for investigators of sexual assault. However, not much documentation exists concerning the examination of a deceased individual with regard to the postmortem interval and presence of such evidence. The authors reviewed cases referred to the medical examiner's office during a 10-year period. During this time, 199 cases were both autopsied and examined for sexual assault. In particular, these examinations included procurement of swabs for Papanicolaou staining of smears and for quantitation of prostatic acid phosphatase. Most of the victims were female, although a few were male. In the majority of cases, the swabs for smears and prostatic acid phosphatase were taken from oral, vaginal, and anorectal areas in females and oral and anorectal areas in males. The smears all were stained with the routine Papanicolaou stain, and intact spermatozoa and spermatozoan heads were sought. The prostatic acid phosphatase was analyzed by the microparticle enzyme immunoassay method and reported as ng/ml. A level of greater than 100 ng/ml was considered positive. The cases were analyzed with respect to postmortem interval; presence or absence of intact spermatozoa or spermatozoan heads; presence of an elevated prostatic acid phosphatase; body location of the specimen; the time of year; location of the victim; and physical injury (anogenital) of sexual assault. The authors hope that by examining the laboratory evidence of sexual assault, a correlation can be drawn between the presence or absence of such evidence and the aforementioned variables.

Significance of antibody to hepatitis B core antigen in blood donors as determined by their serologic response to hepatitis B vaccine.

Because large numbers of volunteer blood donors may be disqualified for "false-positive" results on tests for antibody to hepatitis B core antigen (anti-HBc), a more specific definition of anti-HBc enzyme immunoassay (EIA)-reactive was evaluated, including only those donor samples that were "strongly" reactive (sample-to-cutoff absorbance ratio, < 0.45). Results using this definition and other anti-HBc test methods were compared to the serologic response (antibody to hepatitis B surface antigen [anti-HBsAg]) to hepatitis B vaccination. Fifty-eight volunteer blood donors who had previously been deferred as donors, because of reactive anti-HBc tests (all other blood screening tests were negative, including those for HBsAg and anti-HBsAg) on two occasions, were vaccinated for hepatitis B. It was assumed that an anamnestic response to vaccine indicated past infection with hepatitis B, while a primary response to vaccine indicated lack of past infection. One (2%) of 43 donors with a historically "weak" anti-HBc (reactive absorbance ratio, > or = 0.45) had an anamnestic response to vaccine, compared to 8 (53%) of 15 with historically "strong" anti-HBc (reactive absorbance ratio, < 0.45) (p < 0.005). Anti-HBc testing using the microparticle EIA method also correlated well with hepatitis B vaccination results. The use of a narrower definition of "reactive" for anti-HBc EIA testing yielded much more specific, but slightly less sensitive, results.