Exploring association between MBL2 gene polymorphisms and the occurrence of clinical blackwater fever through a case-control study in Congolese children.

BACKGROUND: Blackwater fever (BWF), one of the most severe and life-threatening forms of falciparum malaria, is characterized by acute massive intravascular haemolysis, often leading to acute renal failure. Thus far, the genetics of the underlying susceptibility to develop BWF is not fully elucidated. Deficiency in the MBL protein, an important component of the innate immune system, has previously been suggested to be a susceptibility factor for the development of severe malaria. This study aimed to evaluate the association between MBL2 gene polymorphisms, known to affect the MBL protein level/activity, and the occurrence of BWF among Congolese children. METHODS: This is a case-control study. Cases were patients with BWF, whereas controls, matched for gender and age, had uncomplicated malaria (UM). Dried blood spot was collected for genotyping. RESULTS: A total of 129 children were screened, including 43 BWF and 86 UM. The common allele in BWF and UM was A, with a frequency of 76.7 and 61.0%, respectively (OR: 2.67 (0.87-829) and p = 0.079). The frequency of the C allele was 18.6 and 29.1% in BWF and UM groups, respectively, with p = 0.858. Not a single D allele was encountered. Genotype AA was at higher risk for BWF whereas genotypes A0 (AB and AC) were over-represented in UM group (OR: 0.21 (0.06-0.78)) with p = 0.019. Nine haplotypes were observed in this study: 3 high MBL expression haplotypes and 6 low MBL expression haplotype. One new haplotype HYPC was observed in this study. None of these haplotypes was significantly associated with BWF. CONCLUSION: This pilot study is a preliminary research on MBL2 gene and infectious diseases in DRC. The study results show a higher risk for BWF in AA. This suggests that future studies on BWF should further investigate the contribution of a strong immune response to the occurrence of BWF.

Induction of two independent immunological cell death signaling following hemoglobinuria -induced acute kidney injury: In vivo study.

The main important clinical signs in acute kidney injury (AKI) after sever Hemiscorpius lepturus envenomation in patients is associated with proteinuria, hemolysis and hemoglobinuria. Unfortunately, our limited knowledge of molecular cell death mechanism in H. lepturus induced AKI restricts the development of desirable therapeutics. So, in the present study, the potential role of necroptosis and ferroptosis in H. lepturus induced AKI were investigated in male albino mice. The animals were administrated by SC injection of venom (1, 2.5, 5 and 10 mg/kg) based on LD50 determination. After 1 and 7 days, urinalysis, stereological assessments and gene expression of Ngal, Tnf-α, Tlr-4, Ripk3, Mlkl and Acsl4 were evaluated by real time PCR. Our data revealed that upregulation of renal Ngal expression is associated with the gene over expression of Tnf-α, Tlr-4, Ripk3 and Mlkl in venom treated kidneys. We observed that the Malondialdehyde (MDA) level was increased in dose-dependent manner similar to Acsl4 gene over expression suggesting a main role of ferroptosis in hemoglobinuria mediated AKI following envenomation. Moreover, transcriptional enhancement of Tlr-4and Tnf-α receptor can cause phosphorylation of Ripk3-Mlkl complex, collapse of membrane potential and DAMPs release which intensified the inflammation cytokines in kidney. Taken together, it supposes co-existence of two separate pathways of regulated necrosis and inflammatory environment provides a promising outlook in prevention and management of hemoglobinuria induced AKI following envenomation in clinical practice.

Quantitative urine test strip reading for leukocyte esterase and hemoglobin peroxidase.

BACKGROUND: Recently, urine test strip readers have become available for automated test strip analysis. We explored the possibilities of the Sysmex UC-3500 automated urine chemistry analyzer based on complementary metal oxide semiconductor (CMOS) sensor technology with regard to accuracy of leukocyte esterase and hemoglobin peroxidase results. We studied the influence of possible confounders on these measurements. METHODS: Reflectance data of leukocyte esterase and hemoglobin peroxidase were measured using CMOS technology on the Sysmex UC-3500 automated urine chemistry analyzer. Analytical performance (imprecision, LOQ) as well as the correlation with white blood cell (WBC) and red blood cell (RBC) counts (Sysmex UF-5000) were studied. Furthermore, the influence of urinary dilution, haptoglobin, pH and ascorbic acid as confounders was determined. RESULTS: Within- and between-run imprecision (reflectance signal) ranged from 1.1% to 3.6% and 0.9% to 4.2% for peroxidase and 0.4% to 2.5% and 0.4% to 3.3% for leukocyte esterase. Good agreement was obtained between the UF-5000 for RBCs and peroxidase reflectance (r=0.843) and for WBCs and leukocyte esterase (r=0.821). Specific esterase activity decreased for WBC counts exceeding 100 cells/µL. Haptoglobin influenced the peroxidase activity, whereas leukocyte esterase and peroxidase activities showed a pH optimum between 5.0 and 6.5. A sigmoidal correlation was observed between urinary osmolality and peroxidase activity. CONCLUSIONS: CMOS technology allows to obtain high quality test strip results for assessing WBC and RBC in urine. Quantitative peroxidase and leukocyte esterase are complementary with flow cytometry and have an added value in urinalysis, which may form a basis for expert system development.

Determination of red blood cells in urinary sediment: Do pH and specific gravity of urine matter?

INTRODUCTION: It has been postulated that erythrocyte cell lysis in urine is common in the case of low urine specific gravity and high urine pH. We aimed to verify the influence of urine dipstick pH and specific gravity on erythrocyturia in the urine sediment in the case of positive dipstick hemoglobinuria. METHODS: We retrospectively collected data on dipstick specific gravity, pH, and urine sediment analysis done by nephrologists in the clinical and research urine laboratory at the Department of Nephrology, University Medical Center Ljubljana. RESULTS: During the 6-year observation period, we analyzed 843 second morning midstream urine samples with positive dipstick hemoglobinuria. Erythrocyturia in urinary sediment was detected significantly less often in urine samples with concomitant hemoglobinuria 1+ and pH < 6.0, (odds ratio (OR) 0.40; 95% confidence interval (CI) 0.21 - 0.76, p = 0.005). The difference was maintained in multivariate analysis including patient age, gender, and specific gravity (OR 0.32; 95% CI 0.15 - 0.65, p = 0.002). In samples with higher grade of hemoglobinuria (≥ 2+), the impact of cell lysis in the case of low pH was negligible. Specific gravity did not have any influence on erythrocyte detection in urinary sediment. CONCLUSIONS: Urinary pH < 6.0 impaired the detection of erythrocytes in urinary sediment, while the dipstick measured urine specific gravity did not have any impact on it. Urine samples with low-grade hemoglobinuria and low pH without erythrocyte detection in urinary sediment should be evaluated again to avoid false-negative results.
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A patient presented with dark brown urine after mothballs powder ingestion.

We report a case who presented with dark brown urine and malaise after mothballs powder ingestion.

Interdependence between urinary cobalt concentrations and hemoglobin levels in pregnant women.

Cobalt is an essential trace element but may cause toxic effects upon occupational or environmental exposure. Women accumulate more cobalt than men at similar exposure levels which may be related to higher metabolic iron loss. During pregnancy these losses are much stronger but their influence on cobalt intake has not been studied. We have studied the associations between changes in hemoglobin and cobalt urinary excretion during pregnancy. 391 pairs of urine and blood samples from pregnant women were collected during the 12th and 32nd weeks of pregnancy and were analyzed for cobalt and hemoglobin. Mean concentrations of urinary cobalt were 0.73 and 1.6 µg/g creatinine during the first and third trimesters, respectively (p<0.001). 84% of pregnant women had higher levels of cobalt in the third than in the first trimester. Cobalt concentrations were negatively associated to hemoglobin levels in the third trimester (p<0.05). Women with higher iron decreases between both trimesters had significant cobalt increases between these two periods. This correspondence involved a statistically significant difference in third trimester mean cobalt concentrations of anemic and non-anemic women, 1.8 and 1.5 µg/g creatinine, respectively (p<0.05). No significant differences between these two groups were found during the first trimester. These results were used to construct generalized additive models both in normal and anemic women. The strong association between the changes of both iron status and cobalt urine levels found in pregnant women may be related to higher intestinal absorption of cobalt at iron depletion such as in the last pregnancy period when iron body demands are high. Possible toxicity effects of these cobalt increases along pregnancy should be considered in cases of populations occupationally or environmentally exposed to this metal.

Acute haemolytic anaemia and myolysis due to G6PD deficiency.

A 2-year-old African-American male patient with sickle cell trait developed cough, red coloured urine, pallor and fatigue. The patient was hospitalised. Diagnostic workup showed that he was glucose 6 phosphate dehydrogenase (G6PD) deficient in erythrocytes. He also had chest X-ray findings of pneumonia. His urine examination showed the presence of haemoglobin and myoglobin. On repeated questioning it was found that he had a moth ball in his mouth a few days prior to this medical episode. This case illustrates a rarely described complication of myolysis in G6PD deficient persons on exposure to a strong oxidant. A review of the literature showed that most people with G6PD deficiency tolerate exercise well without untoward effect in muscles. However, assay of myoglobin in urine has not been routinely performed in these patients during acute haemolytic episode, and thus it is uncertain how frequent myoglobulinaemia occurs in a similar stress situation.

Development of hybrid organic-inorganic surface imprinted Mn-doped ZnS QDs and their application as a sensing material for target proteins.

Applying molecular imprinting techniques to the surface of functionalized quantum dots (QDs) allows the preparation of molecularly imprinted polymers (MIPs) with accessible, surface exposed binding sites and excellent optical properties. This paper demonstrates a new strategy for producing such hybrid organic-inorganic imprinted Mn-doped ZnS QDs for specific recognition of bovine hemoglobin. The technique provides surface grafting imprinting in aqueous solutions using amino modified Mn-doped ZnS QDs as supports, acrylamide and methacrylic acid as functional monomers, γ-methacryloxypropyl trimethoxy silane as the grafting agent, and bovine hemoglobin as a template. The amino propyl functional monomer layer directs the selective occurrence of imprinting polymerization at the QDs surface through copolymerization of grafting agents with functional monomers, but also acts as an assistive monomer to drive the template into the formed polymer shells to create effective recognition sites. Using MIP-QDs composites as a fluorescence sensing material, trace amounts of bovine hemoglobin are signaled with high selectivity by emission intensity changes of Mn-doped ZnS QDs, which is embedded into the imprinted polymers.

Association between urine osmolality and specific gravity in dogs and the effect of commonly measured urine solutes on that association.

OBJECTIVE: To determine the association between urine osmolality and specific gravity (USG) in dogs and to evaluate the effect of commonly measured urine solutes on that association. ANIMALS: 60 dogs evaluated by an internal medicine service. PROCEDURES: From each dog, urine was obtained by cystocentesis and USG was determined with a refractometer. The sample was divided, and one aliquot was sent to a diagnostic laboratory for urinalysis and the other was frozen at -80°C until osmolality was determined. Urine samples were thawed and osmolality was measured in duplicate with a freezing-point depression osmometer. The correlation between mean urine osmolality and USG was determined; the effect of pH, proteinuria, glucosuria, ketonuria, bilirubinuria, and hemoglobinuria on this relationship was investigated with multiple regression analysis. RESULTS: The Pearson correlation coefficient between urine osmolality and USG was 0.87. The final multivariable regression model for urine osmolality included USG and the presence of ketones; ketonuria had a small negative association with urine osmolality. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated a strong linear correlation between osmolality and USG in urine samples obtained from dogs with various pathological conditions, and ketonuria had a small negative effect on that correlation.

Selective room temperature phosphorescence sensing of target protein using Mn-doped ZnS QDs-embedded molecularly imprinted polymer.

The direct correlation between disease states and protein levels makes the sensitive, convenient, and precise detection of proteins the focus of scientific research. This paper demonstrates a new strategy for producing phosphorescent molecularly imprinted polymer (MIP) for specific recognition of a target protein. The technique provides surface graft imprinting in aqueous solutions using vinyl modified Mn-doped ZnS QDs as supports, methacrylic acid and acrylamide as functional monomers, and bovine hemoglobin as a template. The QDs act as antennae for recognition signal amplification and optical readout, and the MIP shell provides analyte selectivity and prevents interfering molecules from coming into contact with the QDs. The small particle sizes and the nontoxicity of the MIP-QDs composites allows for good dispersibility and stability in an aqueous solution. Under optimal conditions, good linear correlations were obtained for bovine hemoglobin over the concentration range from 1.0×10⁻7 to 5.0×10⁻6 mol L⁻¹ and with recoveries of 96.7-103.8% and 92.6-94.2% for urine and serum samples, respectively. The long lifetime of the MIP-QDs composites phosphorescence avoids interference due to autofluorescence and scattering of the biomatrix, facilitating composites' application for detection of bovine hemoglobin in biological fluids.

Sensitive detection of trace hemoglobin using fluorescence method based on functionalized quantum dots.

The fluorescence quenching of quantum dots by hemoglobin has been demonstrated to depend on surface functionalization, and this property has been utilized to construct a novel fluorescent method for rapid, sensitive, and selective detection of trace hemoglobin in urine at microgram level. This method shows low interference and high selectivity for hemoglobin with a limit of detection of 4.3 µg L(-1) in water and 66.1 µg L(-1) in urine, which are lower than those of currently used methods in labs and clinics. Spike and recovery tests in raw, acidified, and alkalized urine samples exhibit good recovery rates for the spiked concentrations close to the limit of detection.

[Evaluation of seasonal changes in hemoglobin A1c in diabetic patients].

In order to evaluate seasonal changes in hemoglobin A1c (HbA1c) values, we examined HbA1c values among 34,590 patients in 2010, and calculated the monthly average of HbA1c values through the year. HbA1c values were the highest in March and the lowest in October with a difference of 0.30%. The similar annual pattern was observed in HbA1c values from 2006 to 2009. Then we selected 453 diabetic patients whose treatment did not change through the year, and calculated average HbA1c values in four seasons each. There were also significant seasonal changes in diabetic patients, which were the highest in the spring and the lowest in the autumn, especially found in patients with insulin therapy. These effects may be caused by cold climate, decreased physical activity, over food intake and body weight gain in the winter. These seasonal changes in HbA1c should be concerned in the case of health service research, clinical trials and evaluation of the effects of medical treatment.

Urinary BTA: indicator of bladder cancer or of hematuria.

BACKGROUND: In this study, we investigated the influence of hematuria on the performance of the bladder tumor antigen (BTA) tests in a clinical cohort and in an experimental model. MATERIALS AND METHODS: Urine samples from a cohort of 126 subjects (64 with BCa and 62 controls) were analyzed by ELISA for hemoglobin and BTA. The experimental model involved the spiking of urine with blood from the same subject, and hemoglobin, red blood cell count, and BTA levels (BTA stat© and BTA-TRAK©). BTA-TRAK© analyses were also performed on serum samples obtained from 40 subjects (20 with confirmed with BCa). RESULTS: In the 126 subject cohort, correlation between hemoglobin and BTA was 0.732. Of the 64 BCa samples, 72 % had a positive BTA assay, but 47 % of controls were also positive. The sensitivity and specificity of BTA to detect BCa was 72 and 53 %, respectively. Hematuria, measured by urinary hemoglobin, was a better indicator of BCa with 75 % sensitivity and 90 % specificity. Spiking of BTA-negative urine samples with as little as 1 µl/10 ml was enough to produce a positive BTA test. High levels of BTA were found equally in the serum of subjects with or without BCa (mean BTA levels 355,159 vs. 332,329 U/ml, respectively). CONCLUSIONS: Rather than detecting a bladder tumor antigen, urinary BTA assays may be measuring serum cFH introduced by bleeding, a common presenting factor in BCa subjects. The presence of hematuria in subjects without malignant disease can result in false-positive BTA assays.

Methodological evaluation and comparison of five urinary albumin measurements.

BACKGROUND: Microalbuminuria is an indicator of kidney damage and a risk factor for the progression kidney disease, cardiovascular disease, and so on. Therefore, accurate and precise measurement of urinary albumin is critical. However, there are no reference measurement procedures and reference materials for urinary albumin. METHODS: Nephelometry, turbidimetry, colloidal gold method, radioimmunoassay, and chemiluminescence immunoassay were performed for methodological evaluation, based on imprecision test, recovery rate, linearity, haemoglobin interference rate, and verified reference interval. Then we tested 40 urine samples from diabetic patients by each method, and compared the result between assays. RESULTS: The results indicate that nephelometry is the method with best analytical performance among the five methods, with an average intraassay coefficient of variation (CV) of 2.6%, an average interassay CV of 1.7%, a mean recovery of 99.6%, a linearity of R=1.00 from 2 to 250 mg/l, and an interference rate of <10% at haemoglobin concentrations of <1.82 g/l. The correlation (r) between assays was from 0.701 to 0.982, and the Bland-Altman plots indicated each assay provided significantly different results from each other. CONCLUSION: Nephelometry is the clinical urinary albumin method with best analytical performance in our study.

Bacillary hemoglobinuria in Japanese black cattle in Hiroshima, Japan: a case study.

Three Japanese Black cows housed with 6 other cows exhibited main clinical symptoms of severe hemoglobinuria. Hematological analyses conducted after antibiotic therapy demonstrated severe anemia, and biochemical analyses indicated both severe hemolysis and disruption of hepatic function. One of the three cows died. Based on the above analyses and observation of typical clinical symptoms, a speculative diagnosis of bacillary hemoglobinuria was made, and immediate high-dose antibiotic treatment improved the general conditions of the surviving animals. Blood samples from the other 2 cows were collected sequentially after antibacterial therapy. Clostridium haemolyticum was detected by a nested polymerase chain reaction analysis of the blood samples. The cows were diagnosed with the second recorded occurrence of bacillary hemoglobinuria in Japan.