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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.

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 LD 50 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

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

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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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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (2014 BMJ Publishing Group Ltd.)

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.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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., (© 2011 Wiley-Liss, Inc.)

Background and Objectives: During Carnival, groups of > or =60 drummers go drumming with their hands and marching for periods of 2 to 4 h. The objective of this study was to determine the frequency and type of urinary abnormalities after candombe drumming and to evaluate possible pathogenic mechanisms., Design, Setting, Participants, & Measurements: For analysis of pathogenic mechanisms, a group of individuals were prospectively evaluated before and after candombe drumming., Methods: Candombe drummers were recruited in January 2006, 1 wk before prolonged drumming. After clinical evaluation, urine and blood samples were obtained before and immediately after drumming., Results: Forty-five healthy individuals (four women and 41 men), median age 31 yr (14 to 56), were evaluated. Predrumming urine and plasma samples were obtained for 30 individuals. Nineteen (42%) of 45 had a previous history of rust urine emission temporally related with candombe drumming. After drumming, 18 of 26 showed urine abnormalities; six of 26 showed rust urine, eight of 26 had microhematuria, and seven of 26 had proteinuria >1 g/L. The candombe drummers who showed rust urine after heavy drumming presented significantly higher levels of lactate dehydrogenase and total bilirubin when compared with those without urine abnormalities. Haptoglobin was significantly lower in the rust urine group. Fragmented red cells were observed in the blood smear of individuals with rust urine. Rust urine after drumming was associated with previous episodes of rust urine and glucosuria., Conclusions: Taken together, these data confirm that rust urine is caused by extracorpuscular hemolysis.

Urinary biomarkers or profiles that allow noninvasive detection of recurrent transitional cell carcinoma (TCC) of the bladder are urgently needed. We obtained duplicate proteomic (SELDI) profiles from 227 subjects (118 TCC, 77 healthy controls and 32 controls with benign urological conditions) and used linear mixed effects models to identify peaks that are differentially expressed between TCC and controls and within TCC subgroups. A Random Forest classifier was trained on 130 profiles to develop an algorithm to predict the presence of TCC in a randomly selected initial test set (n = 54) and an independent validation set (n = 43) several months later. Twenty two peaks were differentially expressed between all TCC and controls (p < 10(-7)). However potential confounding effects of age, sex and analytical run were identified. In an age-matched sub-set, 23 peaks were differentially expressed between TCC and combined benign and healthy controls at the 0.005 significance level. Using the Random Forest classifier, TCC was predicted with 71.7% sensitivity and 62.5% specificity in the initial set and with 78.3% sensitivity and 65.0% specificity in the validation set after 6 months, compared with controls. Several peaks of importance were also identified in the linear mixed effects model. We conclude that SELDI profiling of urine samples can identify patients with TCC with comparable sensitivities and specificities to current tumor marker tests. This is the first time that reproducibility has been demonstrated on an independent test set analyzed several months later. Identification of the relevant peaks may facilitate multiplex marker assay development for detection of recurrent disease.

During a prospective study of red cell variants and severe malaria in children, a surprising observation was the occurrence of dark urine. Children were grouped according to urine findings: 22 had dark urine that contained a haem protein (Group I), 93 had urine of normal colour that contained a haem protein (Group II) and 236 had normal urine (Group III). To investigate the cause of dark urine, haemolysis and muscle cell injury were assessed. Intravascular haemolysis was greater in Group I than in Groups II and III. However, anaemia was more severe in Group III and is likely to have resulted mainly from extravascular haemolysis. Median plasma myoglobin concentrations were greater in Groups I and II than Group III (P = 0.00060). Plasma myoglobin was greater in children with cerebral malaria, hyperlactataemia and those who died but was not associated with acidosis. Urine myoglobin was greater in Group I than Groups II and III (P = 0.00054). It is likely that both haemoglobin and myoglobin contributed to dark urine. The association between muscle cell injury and coma suggests sequestration of parasitized red cells as a common underlying pathology. In malaria, hyperlactataemia may result directly from breakdown of muscle protein as well as tissue hypoxia.

The aim of this study was to evaluate the automated urine test strip analyzer Aution Max AX 4280 which uses strips able to measure 12 urinary parameters. For precision study, we considered glucose, protein, blood, leukocytes, nitrite, and pH and the four first were compared with usual methods: chemical measurement or microscopic examination. Reproducibility of the semi quantitative results was determined as the proportion of results falling into the same concentration range. Within-run reproducibility assessed using urine specimens were between 83% and 96%. Between day on quality controlled materials, it was higher (96%), and leukocytes were shared between two classes 250 and 500 cells/microL. For glucose and proteins, linearity was good and comparison with quantitative methods yielded high correlation. The false negative fraction was low and there was no false positive results. For blood, fraction of false positive was 7.5% for 0.3 mg/L of hemoglobin compared with microscopic analysis and false negative fraction was 1.3%. For leukocytes, false positive fraction was 4%, false negative one was 7.8%. These results, values for sensitivity, specificity, predictive positive value, predictive negative value permitted to determine the cut-off values to realize second levels analysis: proteins are measured from 0.30 g/L, glucose from 2.8 mmol/L, microscopic urinalysis was performed from hemoglobin more than 0.3 mg/L and leukocytes more than 25 cells/microL.

The quantification method of urinary hemoglobin has not been established. We examined whether a reagent (Eiken, Tokyo) used to immunologically assay fecal hemoglobin could be utilized to quantify urinary hemoglobin. The coefficients of variation were 2.2-3.0% in the reproducibility of one-run assays using urine and 4.3-5.7% in that of multiple-run assays using the standard sample of hemoglobin. Urinary hemoglobin was unstable and decreased in a time- and temperature-dependent manner. However, addition of the hemoglobin stabilization buffer (50 mM phosphate buffer, pH 6.4) (Eiken) to urine made urinary hemoglobin stable. Urinary hemoglobin levels did not change significantly when stored at 4 degrees C for 7 days or at -80 degrees C for 30 days. The hemoglobin concentration (mean +/- SD) of urine showing 1+, 2+ and 3+ with a test strip was 385 +/- 165(n = 30), 1070 +/- 499(n = 40) and 4130 +/- 2770 ng/ml (n = 20), respectively. Urinary hemoglobin did not correlate with urinary albumin, transferrin, immunoglobulin G, N-acetyl-beta-D-glucosaminidase nor alpha 1 microglobulin. These results suggest that this immunological method using the hemoglobin stabilization buffer can be utilized for quantification of urinary hemoglobin, which may provide clinically important information.

Objective: To observe whether there are any injuries to muscle and deleterious effects on the liver and kidneys during training and after competition in Thai boxers., Methods: Serum levels of intracellular enzymes and specific markers in the urine were measured during training and after fighting in Thai boxers., Results: During the training period, the activities of muscle enzymes were significantly increased whereas those of the liver enzymes and creatinine clearance were not changed. After a match, on the other hand, both liver and muscle enzyme activities were elevated but renal function was decreased., Conclusions: The training protocol for Thai boxers has virtually no deleterious effect on liver and renal function, but damage to skeletal muscle cells may occur. However, competition may cause muscle injury without any obvious damage to the liver and kidneys.

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of Chlamydia trachomatis nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for C. trachomatis detection. Aliquots were spiked with the equivalent of one C. trachomatis elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%; P = 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odds ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and -70 degreesC overnight or were extracted with phenol-chloroform and then retested at dilutions of 1:1, 1:4, and 1:10. Most inhibition was removed by storage overnight at 4 or -70 degreesC and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors.

The purpose of this study was to determine the effect of the hemoglobin based oxygen carrier, polyethylene glycol conjugated bovine hemoglobin (PEG-Hb) on the physiology of the rat. This study was divided into the following 3 parts: pharmacokinetics, cardiovascular, and histopathology. Pharmacokinetic studies evaluated the PEG-Hb circulatory life and the resultant effect on urine composition. Telemetric intravascular blood pressure probes monitored the heart rate and mean arterial pressure. Renal arterial blood flow was determined by intraoperative perivascular ultrasound. Tissue histology was evaluated for both time and model dependent responses. The mean circulatory half-life of PEG-Hb was 17.7+/-0.3 h. Proteinuria and hemoglobinuria were greatly reduced with PEG conjugation. PEG-Hb treated rats produced 8.5 times and 49 times less proteinuria and hemoglobinuria, respectively, than unmodified bovine Hb treated animals. The mean arterial pressure (MAP) in PEG-Hb treated rats was insignificantly different from sham controls undergoing a 30% exchange transfusion while dextran caused an initial reduction and bovine Hb produced a prolonged elevation in the MAP. In these same anesthetized rats, PEG-Hb slightly decreased the heart rate while dextran caused an increase and bovine Hb had no effect. In addition, PEG-Hb was able to maintain the renal arterial blood flow while both Ringer's lactate and bovine Hb caused a reduction in the blood flow. Finally, PEG-Hb treated rats showed a dose and time dependent formation of vacuoles within the renal proximal convoluted tubules and splenic macrophages in both top-load and exchange transfusion models, but no other morphological changes. In conclusion, PEG-Hb had a relatively long vascular persistence that did not cause any significant alterations in the urinalysis, cardiovascular function, or tissue histopathology in the rat.

This study was designed to determine the effect of polyethylene glycol (PEG) conjugation on stroma-free bovine hemoglobin. This was accomplished by examining the effects of unmodified stroma-free bovine hemoglobin (bovine Hb), PEG modified bovine hemoglobin (PEG-Hb) and dextran 70 on heart rate (HR), mean arterial pressure (MAP), gross renal morphology, blood chemistry, and hemoglobinuria development in conscious beagle dogs following a 30% exchange transfusion. Dogs were implanted with telemetric blood pressure probes and after 2 weeks underwent an isovolumic 30% blood volume exchange transfusion. Dogs treated with bovine Hb displayed a significant increase in MAP for 2 h following the exchange transfusion with no effect on HR. These animals exhibited significant levels of hemoglobinuria (> 20% of infused dose) within 24 h. Histopathologically, all bovine Hb infused dogs displayed renal tubular vacuolization, with 2 dogs showing regions of tubular casts and tubular cell regeneration. PEG-Hb was shown to have a circulatory half-life of 58.3 +/- 2.4 h and caused no significant changes in MAP or HR throughout the study period. Dogs excreted less than 0.1% of infused PEG-Hb within 24 h and displayed only renal tubular epithelial cell vacuolization. Dextran 70 caused a slight but insignificant decrease in MAP, elevated the HR, and exhibited only slight renal vacuole formation. Blood chemistry remained essentially stable following exchange transfusion with all the test articles. The conjugation of PEG to hemoglobin greatly increased the parent protein's vascular retention while attenuating some of its less favorable attributes.

Two cases are described which show that on urine protein electrophoresis a paraprotein owing to hemoglobin cannot be distinguished from a Bence Jones protein. A simple method is described for confirming the presence of hemoglobin: a densitometric scan of the electrophoretogram at 415 nm, in which hemoglobin absorbs in a band coincident with the restriction. Furthermore, it is shown that other serum proteins, elevated levels of bilirubin, and Bence Jones protein do not interfere with this detection system.

To study the effect of cardioprotection type on haemolysis, 100 patients scheduled for elective coronary artery bypass grafting were allocated to receive either blood cardioplegia (BCP) or crystalloid cardioplegia (CCP). Haemoglobin concentrations in plasma and urine were measured after induction of anaesthesia, 1 hour postoperatively and the next morning; blood acid-base status was determined at the end of cardiopulmonary perfusion; serum electrolytes and creatinine were measured before and 1 and 3 hours after the operation and serum creatinine also the next morning. Plasma haemoglobin values tended to be higher in the CCP than in the BCP group (47.6, 200.2 and 69.1 vs 31.5, 207.5 and 39.2 mg/l, p = 0.084). The urinary haemoglobin concentrations did not differ between the groups. The acid-base status showed greater buffer capacity with BCP technique. These results contradict association of blood cardioplegia technique with increased haemolysis during coronary artery bypass grafting.

A 2-year-old spayed female Shetland Sheepdog had recurrent episodes of discolored urine. Treatments administered for presumed urinary tract infection did not prevent recurrence. Episodes of pigmenturia appeared to correlate with stressful situations or excessive activity. Examination of urine sediment consistently revealed that RBC were not evident, despite a positive result for blood on urinalysis. This was suggestive of hemoglobinuria, and diagnostic testing was instituted to determine the underlying cause. Results of alkaline and osmotic fragility tests were useful in determining that an increase in erythrocyte fragility was the underlying cause of the recurrent pigmenturia. Erythrocyte fragility testing should be considered in animals that do not respond to appropriate treatments for pigmenturia.

This report describes the tissue distribution and long-term (14-day) excretion of hemoglobin cross-linked between the alpha-chains (alpha alpha Hb) with carbon 14-labeled bis(3,5-dibromosalicyl)fumarate. Fully conscious, chronically cannulated rats (n = 40) were treated with a 50% isovolemic exchange transfusion (ET) with solutions of 14C-labeled alpha alpha Hb (8.0 gm/dl) and were then monitored for as long as 14 days. Thirteen tissue types were analyzed for radioactivity by liquid scintillation counting. The highest concentration of label was found in the kidney and in tissues of the reticuloendothelial system (i.e., spleen, bone marrow, and liver). The 14C-labeled alpha alpha Hb did not appear to cross the blood-brain barrier, because radioactivity in the brain was barely detectable. The dose of 14C-labeled alpha alpha Hb (2.4 gm Hb/kg) produced an initial plasma Hb level of 4.6 gm/dl, with a half-life in the plasma of 5.0 hours. The peak concentration in kidney, spleen, and liver occurred at 24 hours after ET, when at least 92% of the 14C-labeled alpha alpha Hb in plasma had been cleared. At 48 hours, red casts were seen in a tiny number of renal tubules in some rats. By 14 days, up to 64% of the injected radioactivity had been recovered in urine and about 10% was recovered in feces. Most excretion occurred 24 to 48 hours after ET. This study demonstrated that 2 weeks were required for the metabolic degradation and elimination of a large dose of alpha alpha Hb in rats.

A method is described for estimating plasma and urinary hemoglobin concentrations as low as 3 mg/L. The assay measures at 528 nm the rate of peroxidation of chlorpromazine by hemoglobin and is not affected by ascorbate and bilirubin concentrations up to 500 mumol/L. Results by this method (mean +/- SD: 54.4 +/- 41.6 mg/L; n = 19) correlated well with those by a scanning spectrophotometric method (52.5 +/- 41.6 mg/L; r = 0.96) and a Coulter Instrument method (r = 0.99; Coulter method: 125 +/- 15 g/L; rate method: 122 +/- 15 g/L; n = 10, r = 0.99). The correlation for assays of 20 plasma samples by our method and a tetramethylbenzidine method was also good (r = 0.95) though the latter gave lower results (31.1 +/- 31.6 mg/L) than the chlorpromazine method (50.9 +/- 41.1 mg/L). The chlorpromazine rate method gave an intra- and interday CV of 7.9% and 9.7%, respectively, at a hemoglobin concentration of 31 mg/L.

Signs of glomerular, proximal and distal tubular dysfunction as well as metabolic control were studied in type 1 diabetes mellitus. To that end, the urinary excretion rates of albumin, sodium, phosphate and Tamm-Horsfall protein as well as HbA1c levels were measured in 20 patients with different degrees of diabetic nephropathy (positive Albustix for several years). Eight diabetic patients with short duration of diabetes and without any diabetic complications and 10 apparently healthy subjects were studied for comparison. The HbA1c levels in the three groups were 8.6 +/- 1.2, 5.9 +/- 2.2 and 4.1 +/- 0.4%, respectively (mean +/- SD). Duration of diabetes in the two diabetic groups were 27 +/- 7 and 3 +/- 1 years, respectively. The urinary protein levels were measured by enzyme-linked immunoassays. The fractional clearance of sodium (1.9 +/- 1.9%; p < 0.001) and phosphate (27 +/- 11%; p < 0.01) were increased in patients with diabetic nephropathy compared to diabetic patients without nephropathy (0.6 +/- 0.2 and 16 +/- 4%) and healthy control subjects (0.6 +/- 0.1 and 16 +/- 4%, respectively). Tamm-Horsfall protein excretion rate was decreased in both diabetic groups (15.0x/3.1 and 37.9x/1.9 micrograms/min, geometric mean x/tolerance factor, p < 0.001 and p < 0.05, respectively) compared to the healthy subjects (63.8x/1.3 micrograms/min). Furthermore, patients with diabetic nephropathy had a lower excretion rate of Tamm-Horsfall protein (15.0x/3.1 micrograms/min) compared to patients without signs of nephropathy (37.9x/1.9 micrograms/min, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Unlabelled: The present experiments were done to determine plasma retention, urinary excretion, and acute renal effects of bolus administration (20 mg/100 g), of hemoglobins (human, bovine and porcine) whose tetrameric structure was stabilized by a pseudo-cross-link between the beta-chains. Standard renal clearance studies showed that these preparations had no immediate adverse effects on GFR or ERPF, however, urine flow and electrolyte excretion commonly increased. Plasma disappearance curves best fit a double-exponential and showed components with half-times of 28 +/- 3 and 257 +/- 43 min. A variable fraction, 57 +/- 6%, appeared in the urine, in phase with the early-fast component of plasma disappearance. This in vivo finding of extensive urinary excretion conflicted with sedimentation velocity analysis of these preparations which indicated a homogenous preparation with mean molecular weight of a stable and therefore, unfilterable tetramer. It appears that the glomerular filter selects for permeant species not easily detected by sedimentation velocity analysis. The different species may reflect the presence, within the PXLs of conformational-slow-equilibria among isomeric forms with different degrees of filterability., In Conclusion: It is important, useful and simple to determine urinary excretion as a means to ascertain molecular stability in vivo.

We describe the first nonimmunological assay of albumin in urine with a detection limit of 1 mg/L. The method is simple, rapid, and accurate. It is based on the probe Albumin Blue 670, which becomes highly fluorescent on binding to albumin. An inexpensive diode laser was used as the light source for measurement of laser-induced fluorescence. The assay was coupled to a flow-injection analysis system capable of running 20 samples per hour. The working range was 1-100 mg/L, which covered albumin concentrations found in nonpathological urine and in urine with slightly increased albumin. This range makes prediction of nephropathy possible at an early stage. Other serum proteins and hemoglobin do not interfere. The coefficients of variation were < 4% and < 7% within one day and from day to day, respectively. A correlation coefficient of 0.990 (n = 100) was obtained for comparison with the Behring nephelometric assay.

Examination of 4379 routine urinalysis specimens with dipsticks sensitive to ascorbic acid showed that 22.8% were positive specimens. The mean urinary vitamin C concentration in this population was 2120 mumol/L. There was a high rate of false-negative dipstick results for hemoglobin in patients with vitamin C in the urine. The highest false-negative rates were observed in urine samples containing less than 50 erythrocytes per high-power field. In further experiments when volunteers consumed supplemental oral USP vitamin C at doses of 100, 250, 500, and 1000 mg or vitamin C-containing fruit juices, even the lowest doses of oral vitamin C or juice resulted in sufficient urinary vitamin C to produce false-negative dipstick results in hemoglobin and glucose testing. To prevent potentially dangerous false-negative results, screening urinalysis protocols relying only on dipstick testing should include a check for urinary vitamin C or use a dipstick that is not subject to vitamin C interference.

Plasma half time of unmodified hemoglobin (UHb) and two intramolecularly cross-linked hemoglobins (alpha alpha XL and beta beta XL) was measured in anesthetized rats after an intravenous bolus of 20 mg.100 gm.-1 To rule out the possibility that differences among plasma half times might be caused by differences in acute effects on renal excretory function, glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured simultaneously with plasma half time. Experiments were also done to determine whether higher doses (60 to 100 mg-1.100 gm-1) of these compounds had a delayed effect (48 hours) on GFR or ERPF. Massive urinary excretion of UHb occurred; however, only 1% of the alpha alpha XL and none of the beta beta XL was excreted. Plasma half time of alpha alpha XL and beta beta XL averaged 3.3 hours, or four times longer than UHb. In no case did a decrease in GFR or ERPF occur. Instead, a transient increase in GFR, ERPF, urine flow, and systemic blood pressure was seen. Similar increases occurred after albumin administration, suggesting expansion of vascular volume as the initiating factor. Renal functions at 48 hours after 60 to 100 mg.100 gm-1 of UHb, alpha alpha XL or beta beta XL were not different from control (albumin). Intratubular hemoglobin casts or intravascular precipitates were not evident in acute or 48-hour studies. At 48 hours Perls' staining material was found in one alpha alpha XL specimen at 3 hours after administration. Perls' staining material was present in renal tubule cells in all but the albumin-treated kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

A solid-phase sandwich enzyme immunoassay for the determination of urinary haemoglobin is described. A screening study was performed to establish whether occult haematuria/haemoglobinuria is present in diabetic patients with elevated urinary albumin excretion. Non-insulin-dependent diabetic patients (145) aged 66.5 years +/- 5.6 with a known duration of diabetes of 10.4 years +/- 6.8 were studied. They delivered a first morning urine sample at consecutive outpatient visits. Erythrocytes were lysed by freezing of the urine samples. The patients were categorized into three groups according to urinary albumin concentration (UAC) as measured by radio-immunoassay. Forty-five patients with microalbuminuria i.e. UAC greater than 20- less than or equal to 200 micrograms/ml had an elevated urinary haemoglobin concentration (UHC) of 38.5 micrograms/l compared to 90 patients with normal UAC and a UHC of 7.7 micrograms/l, p less than 10(-4). A further increase was seen in 10 proteinuric patients, UHC 161.8 micrograms/l, p less than 0.05. The urinary concentration of albumin and haemoglobin were significantly correlated, r = 0.49, p less than 10(-8). In 32 insulin-dependent diabetics the findings were similar. There was no correlation between either age or known duration and the haemoglobin concentration. In 42 normal subjects, the UHC was 3.6 micrograms/lx/divided by 6.1. Haematuria has formerly been described in patients with diabetic nephropathy alone. The present findings suggest that occult haematuria/haemoglobinuria is already present in patients with microalbuminuria.

In hematuria, dysmorphic red blood cells found in the urine sediment are known to indicate glomerular origin of bleeding. To better understand the mechanisms which might cause the typical membrane changes in dysmorphic erythrocytes in vivo, we have exposed normal human erythrocytes in vitro to an osmotic and enzymatic environment similar to that of the nephron. In addition, treated or untreated erythrocytes were exposed to a hemolytic environment. The morphology of the cells was evaluated by light and electron microscopy in comparison of that of dysmorphic erythrocytes obtained in vivo. Neither the passage of erythrocytes in sequence through various solutions each simulating the tubule fluid in a different part of the nephron, nor exposure to urinary or renal enzymes or to serum per se causes the typical membrane alterations. Upon exposure of passaged erythrocytes to a hemolytic environment, however, 50 to 90% of the cells become dysmorphic in a time and dose dependent fashion. By light and electron microscopy these dysmorphic erythrocytes obtained in vitro are indistinguishable from those obtained in vivo. In contrast, non-passaged red blood cells are not affected by the same hemolytic environment. We conclude that osmotically challenged erythrocytes exposed to a hemolytic environment develop dysmorphic membrane alterations.

A 16 years old boy had a typical exertional hemoglobinuria after walking with his new leather shoes on. The attacks of hemoglobinuria were associated with the appearance of an unstable hemoglobin in red cells. A slightly decreased fragility curve by 24 hr incubated red cells, the increase of autohemolysis and the appearance of heat labile hemoglobin were observed after exertion. These findings support that exertional hemoglobinuria may be regarded as a transitory erythropathia.

Technical advances in laboratory testing such as sensitive chemical tests and standardized commercial slides have significantly altered our entire approach to urinalysis. In addition, the recognition of the association of microhematuria with urologic neoplasms and the morphologically altered red blood cells with renal parenchymal bleeding is beginning to expand our knowledge of the pathogenesis of microhematuria. Future trends may include a broader range of chemical tests as well as a better understanding of disease processes associated with microhematuria.

Red cell indices were determined in 6 well trained runners before and after a 100 km race, and Coulter Counter (CC) determinations compared with calculated values derived from centrifuged hematocrit (ctrf), red cell count (CC) and hemoglobin measurements. The following changes were observed immediately after the race, as compared to values 3 days before: MCV(ctrf) decreased by 4.9% (p less than 0.001), MCV(CC) increased by 1.9% (p less than 0.05), MCHC(ctrf) increased by 4% and MCHC(CC) decreased by 3%. The increase in MCV(CC) suggests that intraerythrocyte osmolality was increased, this probably leading to swelling of the cells induced by a shift of water from the diluting Coulter Counter solution into the red cells prior to the MCV measurement. The decrease in MCV(ctrf) immediately after the race was not correlated with the increase in plasma osmolality. This suggests that plasma osmolality alone was not the key factor for regulation of red cell volume. The changes in MCV(ctrf), which contributed to a surprising stability of the hematocrit value and plasma volume, might represent a physiological principle for the maintenance of a favourable blood viscosity.

1. Some practical aspects of the assay of urinary N-acetyl-beta-glucosaminidase activity were studied in patients with renal disease. 2. Centrifugation and dialysis of urine were not necessary prior to assay. 3. When enzyme activity was related to urinary creatinine random urine samples could be used. 4. Since preservatives used to prevent bacterial growth may inhibit enzyme activities, samples should be assayed immediately after collection or stored at 4 degrees C or 20 degrees C. 5. Mild visible haemoglobinuria, eg. derived from lysed red blood cells did not interfere with the enzyme assay.