An automated microfluidic chemiluminescence immunoassay platform for quantitative detection of biomarkers.

A rapid, sensitive and quantitative biomarker detection platform is of great importance to the small clinic or point-of-care (POC) diagnosis. In this work, we realize that an automated diagnostic platform mainly includes two components: (1) an instrument that can complete all steps of the chemiluminescence immunoassay automatically and (2) an integrated microfluidic chip which is disposable and harmless. In the instrument, we adopt vacuum suction cups which are driven by linear motor to realize a simple, effective and convenient control. The method of acridine esterification chemiluminescence is adopted to achieve a quantitative detection, and a photomultiplier tube is used to detect photons from acridine ester producing in alkaline conditions. We use the laser cutting machine and hot press machine to accomplish the product of microfluidic chips. The automated microfluidics-based system is demonstrated by implementation of a chemiluminescence immunoassay for quantitative detection of ferritin. We observe alinear relationship between CL intensity and the concentration of ferritin from 5.1 to 1300 ng mL -1and the limit of detection (LoD) is 2.55 ng mL -1. At the same time, we also used the automated microfluidics-based system to test clinical serum samples. The whole process of chemiluminescence experiment can complete within 45 min. We realize that this lab-on-a-chip chemiluminescence immunoassay platform with features of automation and quantitation provides a promising strategy for POC diagnosis.

ASSESSMENT OF DRUSEN AND OTHER RETINAL DEGENERATIVE CHANGES IN PATIENTS WITH HEREDITARY HEMOCHROMATOSIS.

PURPOSE: Iron can exert oxidative damage, and increased accumulation is believed to play a role in age-related macular degeneration. Hereditary hemochromatosis leads to an increase in total body iron. Patients with HH were assessed for drusen and other retinal changes. METHODS: Descriptive uncontrolled study of spectral-domain optical coherence tomography, short-wavelength autofluorescence, and color fundus images from patients with HH were used. Diagnosis of HH was established by measuring ferritin and transferrin saturation, and confirmed by genetic testing. Classification of the patients according to initial ferritin level was: Group A >1,032 µg/L; Group B below. RESULTS: Twenty-five percent of the invited participants were enrolled. Mean age at diagnosis was 46 ± 15 years in Group A, and 38 ± 13 years in Group B, P = 0.07, whereas mean age at imaging was 60 ± 13 years in Group A, and 48 ± 15 years in Group B (P = 0.003). The median of the initial ferritin level was 1,869 (1,262-3,256) ng/mL in Group A, and 534 (439-679) ng/mL in Group B. No subject in either group revealed multiple drusen, unambiguous changes of the retinal pigment epithelium, or increased lipofuscin in any of the images. CONCLUSION: The study results did not show an increased prevalence of drusen or other retinal degenerative changes in patients with HH. Thus, it was concluded that increased intestinal iron absorption as well as increased blood iron concentration are not risk factors for the early development of retinal degenerative changes in this study population.

Upfront dilution of ferritin samples to reduce hook effect, improve turnaround time and reduce costs.

INTRODUCTION: Sandwich immunoassays offer advantages in the clinical laboratory but can yield erroneously low results due to hook (prozone) effect, especially with analytes whose concentrations span several orders of magnitude such as ferritin. This study investigated a new approach to reduce the likelihood of hook effect in ferritin immunoassays by performing upfront, five-fold dilutions of all samples for ferritin analysis. The impact of this change on turnaround time and costs were also investigated. MATERIALS AND METHODS: Ferritin concentrations were analysed in routine clinical practice with and without upfront dilutions on Siemens Centaur® XP (Siemens Healthineers, Erlang, Germany) immunoanalysers. In addition, one month of baseline data (1026 results) were collected prior to implementing upfront dilutions and one month of data (1033 results) were collected after implementation. RESULTS: Without upfront dilutions, hook effect was observed in samples with ferritin concentrations as low as 86,028 µg/L. With upfront dilutions, samples with ferritin concentrations as high as 126,050 µg/L yielded values greater than the measurement interval and would have been diluted until an accurate value was obtained. The implementation of upfront dilution of ferritin samples led to a decrease in turnaround time from a median of 2 hours and 3 minutes to 1 hour and 18 minutes (P = 0.002). CONCLUSIONS: Implementation of upfront dilutions of all ferritin samples reduced the possibility of hook effect, improved turnaround time and saved the cost of performing additional dilutions.

Assessment of ferritin content in multiple sclerosis brains using temperature-induced R*2 changes.

PURPOSE: Current MRI techniques cannot reliably assess iron content in white matter due to the confounding diamagnetic effect of myelin. The purpose of this study was to validate with histology a novel iron mapping technique that uses the temperature dependency of the paramagnetic susceptibility in multiple sclerosis (MS) brains, where white matter has been reported to show significant variations in iron content. METHODS: We investigated post mortem brain tissue from three MS patients and one control subject. Temperature-dependent R2* relaxometry was performed between 4°C and 37°C. The resulting temperature coefficient ( TcR2*) maps were compared with immunohistochemical stains for ferritin light chain. RESULTS: Good agreement between TcR2* maps and ferritin staining was found by way of visual comparison and quantitative analysis. The highest iron concentrations were detected at the edge of MS lesions and in the basal ganglia. For all regions, except the subcortical U-fibers, there was a significant negative correlation between the TcR2* values and the ferritin count. CONCLUSION: This study provides further evidence that TcR2* may be a reliable measure of white matter iron content due to the elimination of myelin-induced susceptibility changes and is well suited for further research into neurological diseases with distortions of the iron homeostasis. Magn Reson Med 79:1609-1615, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

The Prevalence and Management of Anemia in Chronic Kidney Disease Patients: Result from the KoreaN Cohort Study for Outcomes in Patients With Chronic Kidney Disease (KNOW-CKD).

Anemia is a common and significant complication of chronic kidney disease (CKD). However, its prevalence and current management status has not been studied thoroughly in Korea. We examined the prevalence of anemia, its association with clinical and laboratory factors, and utilization of iron agents and erythropoiesis stimulating agents using the baseline data from the large-scale CKD cohort in Korea. We defined anemia when hemoglobin level was lower than 13.0 g/dL in males and 12.0 g/dL in females, or received by erythropoiesis stimulating agents. Overall prevalence of anemia was 45.0% among 2,198 non-dialysis CKD patients from stage 1 to 5. Diabetic nephropathy (DN) as a cause, CKD stages, body mass index (BMI), smoking, leukocyte count, serum albumin, iron markers, calcium, and phosphorus concentration were identified as independent risk factors for anemia. Considering the current coverage of Korean National Health Insurance System, only 7.9% among applicable patients were managed by intravenous iron agents, and 42.7% were managed by erythropoiesis stimulating agents.

Effects of menopause on blood manganese levels in women: analysis of 2008-2009 Korean National Health and Nutrition Examination Survey data.

INTRODUCTION: We present data from the Korean National Health and Nutrition Examination Survey 2008-2009 on the association between blood manganese (Mn) levels and menopausal status in women. METHODS: The present analysis was restricted to female participants who completed the health examination survey, including blood Mn measurements (n=1826). Menopausal status was categorized into pre- and post-menopausal. Multivariable linear regression analyses were performed to determine whether menopausal status or serum ferritin were significant predictors of blood Mn level and to investigate whether menopausal status modifies the association between blood Mn and serum ferritin after adjusting for covariates. RESULTS: The geometric means (GMs) of blood Mn in the low and low-normal serum ferritin groups were significantly higher than the GM of blood Mn in the normal group. The GM of blood Mn in premenopausal women was significantly higher than in postmenopausal women Multivariable linear regression analyses showed that both serum ferritin and menopausal status were predictors of blood Mn, after adjusting for various covariates, and menopausal status acted as a modifier of the effect of ferritin on blood Mn levels. Blood Mn levels were 11.0% and 22.7% lower in premenopausal women when serum ferritin increased from 10 µg/dL to 60 and 100 µg/dL, respectively, but the decrease in blood Mn based on the increase in serum ferritin was minimal in postmenopausal women. DISCUSSION: To our knowledge, this is the first report that menopausal status was a predictor of blood Mn level after adjusting for serum ferritin. In conclusion, the present study showed that both serum ferritin and menopausal status were predictors of blood Mn, after adjusting for various covariates, and menopausal status acted as a modifier of the effect of ferritin on blood Mn levels.

Transient elastography in the assessment of liver fibrosis in adult thalassemia patients.

Transient elastography (TE) is a valuable noninvasive technique of measuring liver stiffness and a reliable tool for predicting hepatic fibrosis in patients with chronic liver disease. The role of TE in patients with beta-thalassemia has not been extensively investigated. The present study aimed to evaluate the role of TE in the assessment of hepatic fibrosis in 115 adult patients with beta-thalassemia major (TM) (#59) or intermedia (TI) (#56). TE was performed according to current practice. Histologic data were obtained in 14 cases. Liver iron concentration was assessed by atomic absorption spectrometry and T2 magnetic resonance. In patients with TM, the proportion of anti-HCV positive viremic patients, median serum ferritin levels, and TE values were significantly higher than in TI. In the group of 14 patients who underwent liver biopsy, a significant positive correlation was observed between liver stiffness and fibrosis stage (r = 0.73, P = 0.003). Severe fibrosis is diagnosed with a sensitivity of 60% and a specificity of 89%, whereas cirrhosis is detected with a sensitivity of 100% and a specificity of 92%. At multivariate analysis, the variables independently associated with TE were ALT, GGT, and bilirubin levels in both groups and, in patients with TM, HCV RNA positivity. In beta-thalassemia patients, TE is a reliable tool for assessing liver fibrosis even if the influence of iron overload has to be clarified.

Combined measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein by an inexpensive, sensitive, and simple sandwich enzyme-linked immunosorbent assay technique.

The measurement of vitamin A (VA) and iron status is very important in the assessment of nutritional deficiencies. The objective of this research was to develop a sandwich ELISA technique for the simultaneous measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein (CRP) as indicators for VA and iron status. The inclusion of CRP as marker of infection allows for more accurate interpretation of VA and iron status. This is accomplished in a 30-microL serum or plasma sample using an ELISA with different capture and detection antibodies and different dilutions of the sample. Commercially available clinical serum controls were used for calibration purposes. The developed assays were compared to commercially available traditional tests. Regression coefficients comparing both assays were better than 0.84 (P < 0.001). Using a limited sample set, the sandwich ELISA assay produced very similar specificity and sensitivity compared to traditional methods when common cutoff values were applied. Intra- and interassay variability was between 5 and 14% for all tests. The cost of the materials for all 5 measurements decreases to less than $1/sample if a large number of samples is analyzed. Due to the low cost, high throughput, and comparability to traditional tests, this procedure has several advantages for assessing VA and iron status in population surveys.

Screening for hemochromatosis: high prevalence and low morbidity in an unselected population of 65,238 persons.

BACKGROUND: Hereditary hemochromatosis (HH) is a common genetic disease leading to accumulation of iron in several organs, most notably the liver. The C282Y/C282Y mutation in the HFE gene is found in most cases. In order to prevent clinical disease and to study the cost and feasibility of screening, a large population was screened. METHODS: In a Norwegian county, all inhabitants 20 years or older were invited to participate in a population-based health survey programme. Screening for HH was one of several subprojects. Blood samples were obtained from 65,238 persons. Subjects with high serum transferrin saturation in two tests and high serum ferritin were clinically evaluated for HH. All subjects with high serum transferrin saturation in two tests were offered genotyping. RESULTS: HH was newly diagnosed in 92 women and 177 men. Phlebotomy treatment was performed in 64 women and 152 men. Severe organ damage (liver cirrhosis) was ascertained in only 4 men. We found no correlation between serum ferritin and age. The estimated cost was US$ 1.6 per subject screened and US$ 390 per newly discovered HH subject. The estimated prevalence of phenotypical HH not previously known was 0.34% in women and 0.68% in men. The prevalence of the C282Y/C282Y mutation was at least 0.68%. CONCLUSION: Large-scale screening for HH can be performed at a relatively low cost if combined with a health survey programme. The yield in terms of newly discovered cases is considerable, but few cases were found seriously ill. Better knowledge of the natural course of HH is necessary if we are to be able to estimate the cost-effectiveness of large-scale screening.

An assessment of asbestos body formation in extrapulmonary sites: liver and spleen.

STUDY OBJECTIVES: Asbestos bodies (ABs) form as asbestos fibers become coated by a cellular iron- and protein-rich matrix. ABs have been reported in lymph nodes and a few extrapulmonary sites, but no data exist as to their formation outside of the lung. It is not clear whether the AB found in these extrapulmonary areas have been transported as mature structures from the lung or formed at the extrapulmonary site. This study was designed to determine if ABs are produced in extrapulmonary sites. The guinea pig efficiently forms ferruginous bodies in the lung and so it was chosen as a model to test the coating efficiency of amosite asbestos fibers in lung, liver and spleen. DESIGN: Sized amosite asbestos (5 mg) was administered either endotracheally into lung (n = 2) or directly into liver (n = 4) and spleen (n = 4) of healthy 10-week-old male guinea pigs. The lung, liver and splenic tissues were removed at 40 and 180 days post inoculation and were examined histologically for the presence of AB via light microscopy. Uncoated fibers isolated from the tissues were characterized by electron microscopy. The coating efficiency was calculated as a ratio of uncoated/coated fibers per organ. RESULTS: The coating efficiency ratios of fibers that were collected at 40 days post-injection from the individual sites were: lung - 350:1, liver - 4200:1, and spleen - 220,000:1. At 6 months post-injection the ratios for the individual sites consisted of: lung - 176:1, liver - 11,000:1, and spleen - 1000:1. CONCLUSION: This study indicates that AB can be formed in extrapulmonary sites and that the coating efficiency in the lung is much greater than that within the liver or spleen.

Total iron-binding capacity-estimated transferrin correlates with the nutritional subjective global assessment in hemodialysis patients.

We examined the value of transferrin concentrations in estimating nutritional status as determined by the subjective global assessment (SGA) score. Fifty-nine hemodialysis patients (37 men and 22 women, aged 59+/-16 years, dialyzed for 3.6+/-3.9 years) were selected by predetermined criteria. All received erythropoietin (EPO) and oral iron therapy. SGA evaluation was conducted twice by both a dietitian and a physician. Serum iron, total iron-binding capacity (TIBC; which is linearly correlated with transferrin), transferrin saturation ratio, ferritin, albumin, total protein, and cholesterol were measured. Twenty-seven (46%) patients were well nourished (group A), 20 (34%) were moderately nourished (group B), and 12 (20%) were poorly nourished (group C) according to the SGA. TIBC values were 276+/-47 mg/dL, 217+/-54 mg/dL, and 176+/-41 mg/dL, respectively (P < 0.00001), and thus directly correlated with the state of nutrition. The relationship between TIBC and nutritional status was independent of age and number of years on hemodialysis. Serum ferritin values were 104+/-93 ng/mL, 161+/-154 ng/mL, and 363+/-305 ng/mL, respectively (P < 0.0003), and thus inversely correlated with the state of nutrition. Transferrin saturation ratios were slightly higher in the severely malnourished patients. The number of years on dialysis were a determinant of nutritional status. These values were 2.4+/-2.4 years for group A, 3.9+/-4.0 years for group B, and 5.7+/-3.9 years for group C (P < 0.05). The average age of the poorly nourished patients was 10 years older than the well-nourished patients. Serum iron values were lower but transferrin saturation ratios were higher in the severely malnourished patients. The required EPO doses were higher in the poorly nourished patients. We suggest that transferrin values are superior to other laboratory tests in assessing nutrition and will supplement SGA criteria. Serum ferritin may be useful as a predictor of illness. Older patients who have been on dialysis longer warrant special concern. Malnutrition may be an indicator of EPO resistance in dialysis patients. Finally, since a decreased TIBC level in poorly nourished patients may erroneously increase the transferrin saturation ratio, our findings may have implications in making the diagnosis and treatment of anemia and iron deficiency in malnourished dialysis patients.

Screening for hemochromatosis in children of homozygotes: prevalence and cost-effectiveness.

Although hereditary hemochromatosis is an autosomal recessive disease, most homozygotes are concerned with the genetic implications for their children. The optimal age for testing children and the cost implications of screening their children have not been clearly established. A clinical database consisting of 255 children from families with at least one homozygote is used to assess the prevalence of homozygotes among children of homozygous parents and to review the biochemical abnormalities and life-threatening symptoms in these young adults. Decision analysis is used to estimate the cost and utility of screening children of a homozygous parent. Eleven homozygotes were discovered among children of homozygotes. Only one male had a life-threatening event, cirrhosis. Decision analysis estimated cost saving of $12 per child screened ($ net present value) and a saving of 10 quality-adjusted days per child screened at age 10 years compared with not screening. If screening began at age 20 years, there is a cost saving of $65 per child screened. Sensitivity analysis showed that the major factors influencing cost savings were the cost of venesections, sensitivity and specificity of the screening tests, and prevalence of disease. Because the prevalence of hemochromatosis is higher in children of homozygotes than in the general population, screening with transferrin saturation and ferritin as early as age 10 years is recommended. Savings are augmented if the cost per venesection is eliminated by allowing hemochromatosis patients to become voluntary blood donors.

Usefulness of serum ferritin levels in the assessment of iron status in non-pregnant Zairean women of childbearing age.

We studied the usefulness of serum ferritin for the assessment of iron deficiency (ID) or ID anaemia (IDA) of 186 lactating and 27 non-lactating Zairean women (15-45 years old). Haemoglobin (Hb), serum iron (SI), total iron binding capacity (TIBC), and transferrin saturation (TS) were also measured. Participants were recruited in rural Bas-Zaire State in the summers of 1986 and 1989. Serum ferritin ranged from 10 to 360 micrograms l-1 (median 62 micrograms l-1) in lactating women and from 14.2 to 120 micrograms l-1 (median 40 micrograms l-1) in non-lactating women. While mean levels of serum ferritin and TS were within the normal range in both groups of women, those of Hb were below normal (< 12 g dl-1), partly due to inflammation. The prevalence of anaemia was 66% in lactating women and 70% in non-lactating women, and did not change with time. It was higher in women with inflammation than in those without inflammation. Although ID (ferritin < 12 micrograms l-1) was almost absent, after raising the cut-off point of ferritin to 50 micrograms l-1 in women with inflammation, it was present in 28.8% of lactating women and 52% of non-lactating women. While the prevalence of ID assessed by serum ferritin was significantly higher in lactating women studied in 1989 (40.5%) than in those studied in 1986 (13.5%), it was similar in both groups of non-lactating women. ID defined by TS < 16% was present in 41% of lactating women and 21% of non-lactating women. In the presence as well as absence of inflammation, the use of TS identified a higher percentage of lactating women with either ID or IDA than did the use of serum ferritin. We conclude that, in the studied population, unless inflammation is taken into consideration, serum ferritin has a limited value in the diagnosis of ID.

PIP: This cross-sectional study involved 213 healthy Zairian women, 15-45 years old, 186 lactating and 27 nonpregnant, nonlactating women and studied the usefulness of serum ferritin levels for the assessment of iron deficiency (ID) or ID anaemia (IDA). Hemoglobin (Hb), serum iron (SI), total iron binding capacity (TIBC), and transferrin saturation (TS) were also measured. The median parity of lactating women was three, and that of nonlactating women was two. They were recruited at the Nsundi Lutete Hospital (Bas-Zaire State) during August and September of 1986 and 1989. Serum ferritin ranged from 10 to 360 mcg 1-1 (median 62 mcg 1-1) in lactating women and from 14.2 to 120 mcg 1-l (median 40 mcg 1-l) in nonlactating women. While mean levels of serum ferritin and TS were within the normal range in both groups of women, those of Hb were below normal ( 12 g dl-1), partly owing to inflammation. The prevalence of anemia was 66% in lactating women and 70% in nonlactating women, and did not change with time. It was higher in women with inflammation than in those without inflammation. Although ID (ferritin 12 mcg 1-1 ) was almost absent, after raising the cut-off point of ferritin to 50 mcg 1-1 in women with inflammation, it was present in 28.8% of lactating women and 52% of nonlactating women. While the prevalence of ID assessed by serum ferritin was significantly higher in lactating women studied in 1989 (40.5%) than in those studied in 1986 (13.5%), it was similar in both groups of nonlactating women. ID defined by TS 16% was present in 41% of lactating women and 21% of nonlactating women. In the presence as well as absence of inflammation, the use of TS identified a higher percentage of lactating women with either ID or IDA than did the use of serum ferritin. In the studied population, unless inflammation is taken into consideration, serum ferritin has a limited value in the diagnosis of ID.

Assessment of hepatic iron overload in thalassemic patients by magnetic resonance spectroscopy.

The transverse relaxation time of water protons is shortened by the presence of iron. This shortening depends on the amount and the environment of iron in the sample. We have developed a method for measuring short transverse relaxation time noninvasively by magnetic resonance spectroscopy. To evaluate magnetic resonance spectroscopy as a means of assessing hepatic iron content in patients with transfusional iron overload, we compared the results obtained with this method with those obtained by other means of assessing total body iron content. The correlation between the liver biopsy iron concentration and 1/transverse relaxation time was highly significant (r = 0.95, p < 0.004, n = 6) for iron loads up to 3% dry weight. The correlation between serum ferritin and 1/transverse relaxation time was also significant, but the correlation coefficient was much lower (r = 0.67, p < 0.002, n = 20). The correlation between 24-hr urinary iron excretion and 1/transverse relaxation time was not significant, nor was that between AST and 1/transverse relaxation time. We conclude that magnetic resonance spectroscopic determination of the transverse relaxation time of hepatic water is an accurate method of measuring liver iron content, especially when the iron content is below 3%. Because it is a noninvasive method that is associated with negligible side effects, it could provide clinicians with an excellent means of assessing the effectiveness of the various therapeutic strategies used in the management of patients with iron overload.

An assessment of the incidence of iron deficiency in paediatric otolaryngology inpatients.

The aims of this study were: to determine whether there is an increased incidence of iron deficiency in paediatric otolaryngology inpatients compared with other surgical controls; and to establish whether preoperative screening of haemoglobin level is warranted in such patients. Children aged 1-10 years admitted electively for ENT surgery or for general surgical procedures had blood taken for haemoglobin level, mean cell volume and serum ferritin. Their age, weight, socioeconomic class and ethnic background were recorded. A total of 100 patients entered the study, in a six-month period. The mean ages and weights for the two groups were statistically different, so allowance was made for this in calculations. Social class was not significantly different. No relationship could be established between haemoglobin level and ferritin level for individual patients. Multiple regression analysis for haemoglobin level, mean cell volume and for ferritin level showed that allowing for the age and weight differences these variables were not significantly different for the two groups. This study has therefore shown no increased incidence of iron deficiency in paediatric ENT inpatients. Each Department should formulate its own policy on pre-operative haemoglobin screening, based on local considerations.

Assessment of the role of nonheme-iron availability in iron balance.

To assess the nutritional relevance of absorption studies that use extrinsically labeled single meals, we developed a method for measuring nonheme-iron absorption from the diet and compared the results with absorption from single meals. When subjects consumed their usual diet, there was good agreement between dietary absorption (6.4%) and representative single meals fed in the laboratory (6.1%). Nonheme-iron availability, as estimated by a model that incorporated the effect of both enhancers and inhibitors, correlated significantly with absorption from single meals but not with dietary absorption. When the diet was modified to promote iron absorption maximally, dietary absorption increased only slightly (8.0%) and remained significantly lower than it was from single meals (13.5%). With an inhibitory diet, the decrease in absorption from single meals was similarly exaggerated. These results indicate that in the context of a varied Western diet, nonheme-iron bioavailability is less important than absorption studies with single meals would suggest.