Free Hemoglobin In Serum Research Articles

Free hemoglobin reference intervals detected with hemoglobin-cyanide method on a biochemical automatic analyzer ILAB 300 PLUS

Objectives - to evaluate the reference intervals for free hemoglobin concentration with the help of hemoglobin-cyanide method and a biochemical automatic analyzer. Material and methods. The reference group consisted of 120 healthy men and 120 healthy women from the Astrakhan region aged from 20 to 60 years. The fulfilment of standard procedures during the pre-analytical prelaboratory, pre-analytical laboratory, analytical and post-analytical stages of the study ensured the quality control for free hemoglobin evaluation. Blood samples were taken with cubital vein puncture after applying a tourniquet (for not more than 1 minute), patient in lying position, using two-component blood sampling systems - disposable polypropylene tubes with a blood coagulation activator (Sarstedt, Germany). To obtain blood serum, blood tubes were centrifuged at 2500 rpm during 10 minutes. The free hemoglobin in blood serum was detected by the hemoglobin-cyanide method using a ILAB 300 PLUS biochemical automatic analyzer (Laboratory Instrumentation, USA). The data was processed using the software package Statistica 6.0 for Windows (StatSoft Inc, США). Results. We used the classical approach and followed the strict inclusion and exclusion criteria, recommended by CLSI C 28-A3 guidelines. Gender and age differences in the content of free hemoglobin in blood serum in men and women of the Astrakhan region were not found. The reference interval of free hemoglobin in men and women of the Astrakhan region was evaluated as 0.02-0.84 g/l.

Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis.

Background A major cause of disability in secondary progressive multiple sclerosis (SPMS) is progressive brain atrophy, whose pathogenesis is not fully understood. The objective of this study was to identify protein biomarkers of brain atrophy in SPMS. Methods We used surface-enhanced laser desorption-ionization time-of-flight mass spectrometry to carry out an unbiased search for serum proteins whose concentration correlated with the rate of brain atrophy, measured by serial MRI scans over a 2-year period in a well-characterized cohort of 140 patients with SPMS. Protein species were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Results There was a significant (p<0.004) correlation between the rate of brain atrophy and a rise in the concentration of proteins at 15.1 kDa and 15.9 kDa in the serum. Tandem mass spectrometry identified these proteins as alpha-haemoglobin and beta-haemoglobin, respectively. The abnormal concentration of free serum haemoglobin was confirmed by ELISA (p<0.001). The serum lactate dehydrogenase activity was also highly significantly raised (p<10-12) in patients with secondary progressive multiple sclerosis. Conclusions An underlying low-grade chronic intravascular haemolysis is a potential source of the iron whose deposition along blood vessels in multiple sclerosis plaques contributes to the neurodegeneration and consequent brain atrophy seen in progressive disease. Chelators of free serum iron will be ineffective in preventing this neurodegeneration, because the iron (Fe2+) is chelated by haemoglobin.

Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis

Background: A major cause of disability in secondary progressive multiple sclerosis (SPMS) is progressive brain atrophy, whose pathogenesis is not fully understood. The objective of this study was to identify protein biomarkers of brain atrophy in SPMS. Methods: We used surface-enhanced laser desorption-ionization time-of-flight mass spectrometry to carry out an unbiased search for serum proteins whose concentration correlated with the rate of brain atrophy, measured by serial MRI scans over a 2-year period in a well-characterized cohort of 140 patients with SPMS. Protein species were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Results: There was a significant (p<0.004) correlation between the rate of brain atrophy and a rise in the concentration of proteins at 15.1 kDa and 15.9 kDa in the serum. Tandem mass spectrometry identified these proteins as alpha-haemoglobin and beta-haemoglobin, respectively. The abnormal concentration of free serum haemoglobin was confirmed by ELISA (p<0.001). The serum lactate dehydrogenase activity was also highly significantly raised (p<10 -12) in patients with secondary progressive multiple sclerosis. Conclusions: The results are consistent with the following hypothesis. In progressive multiple sclerosis, low-grade chronic intravascular haemolysis releases haemoglobin into the serum; the haemoglobin is subsequently translocated into the central nervous system (CNS) across the damaged blood-brain barrier. In the CNS, the haemoglobin and its breakdown products, including haem and iron, contribute to the neurodegeneration and consequent brain atrophy seen in progressive disease. We postulate that haemoglobin is a source of the iron whose deposition along blood vessels in multiple sclerosis plaques is associated with neurodegeneration. If so, then chelators of haemoglobin, rather than chelators of free serum iron, may be effective in preventing this neurodegeneration.

Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis

Background: A major cause of disability in secondary progressive multiple sclerosis (SPMS) is progressive brain atrophy, whose pathogenesis is not fully understood. The objective of this study was to identify protein biomarkers of brain atrophy in SPMS. Methods: We used surface-enhanced laser desorption-ionization time-of-flight mass spectrometry to carry out an unbiased search for serum proteins whose concentration correlated with the rate of brain atrophy, measured by serial MRI scans over a 2-year period in a well-characterized cohort of 140 patients with SPMS. Protein species were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Results: There was a significant (p&lt;0.004) correlation between the rate of brain atrophy and a rise in the concentration of proteins at 15.1 kDa and 15.9 kDa in the serum. Tandem mass spectrometry identified these proteins as alpha-haemoglobin and beta-haemoglobin, respectively. The abnormal concentration of free serum haemoglobin was confirmed by ELISA (p&lt;0.001). The serum lactate dehydrogenase activity was also highly significantly raised (p&lt;10-12) in patients with secondary progressive multiple sclerosis. Conclusions: An underlying low-grade chronic intravascular haemolysis is a potential source of the iron whose deposition along blood vessels in multiple sclerosis plaques contributes to the neurodegeneration and consequent brain atrophy seen in progressive disease. Chelators of free serum iron will be ineffective in preventing this neurodegeneration, because the iron (Fe2+) is chelated by haemoglobin.

Human mannose-binding lectin inhibitor prevents Shiga toxin–induced renal injury

Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in STEC HUS. Using novel human MBL2-expressing mice (MBL2 KI) that lack murine Mbls (MBL2(+/+)Mbl1(-/-)Mbl2(-/-)), anovel STEC HUS model consisted of an intraperitoneal injection with Shiga toxin-2 (Stx-2) with or without anti-MBL2 antibody (3F8, intraperitoneal). Stx-2 induced weight loss, anemia, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, butasignificantly decreased glomerular filtration rate compared with control/sham mice. Immunohistochemical staining revealed renal C3d deposition and fibrin deposition inglomeruli in Stx-2-injected mice. Treatment with 3F8 completely inhibited serum MBL2 levels and significantly attenuated Stx-2 induced-renal injury, free serum hemoglobin levels, renal C3d, and fibrin deposition and preserved the glomerular filtration rate. Thus, MBL2 inhibition significantly protected against complement activation and renal injury induced by Stx-2. This novel mouse model can beused to study the role of complement, particularly lectin pathway-mediated complement activation, in Stx-2-induced renal injury.

Detecting free hemoglobin in blood plasma and serum with luminescent terbium complexes.

Hemolysis, the rupturing of red blood cells, can result from numerous medical conditions (in vivo) or occur after collecting blood specimen or extracting plasma and serum out of whole blood (in vitro). In clinical laboratory practice, hemolysis can be a serious problem due to its potential to bias detection of various analytes or biomarkers. Here we present the first "mix-and-measure" method to assess the degree of hemolysis in biosamples using luminescence spectroscopy. Luminescent terbium complexes (LTC) were studied in the presence of free hemoglobin (Hb) as indicators for hemolysis in TRIS-buffer, and in fresh human plasma with absorption, excitation and emission measurements. Our findings indicate dynamic as well as resonance energy transfer (FRET) between the LTC and the porphyrin ligand of hemoglobin. This transfer leads to a decrease in luminescence intensity and decay time even at nanomolar hemoglobin concentrations either in buffer or plasma. Luminescent terbium complexes are very sensitive to free hemoglobin in buffer and blood plasma. Due to the instant change in luminescence properties of the LTC in presence of Hb it is possible to access the concentration of hemoglobin via spectroscopic methods without incubation time or further treatment of the sample thus enabling a rapid and sensitive detection of hemolysis in clinical diagnostics.

Naturally occurring alpha-synuclein autoantibodies in Parkinson's disease: sources of (error) variance in biomarker assays.

Alpha-synuclein (α-Syn) plays a pivotal role in the pathophysiology of Parkinson’s disease (PD), which can partly be modulated by innate and adaptive immune functions, and vice versa. Here, naturally occurring α-Syn autoantibodies (α-Syn-nAbs) may be effective against α-Syn pathoetiology and may serve as a PD biomarker. However, serum and cerebrospinal fluid α-Syn-nAbs levels still lack consistent evidence as required for a reliable PD biomarker. Serum and cerebrospinal fluid α-Syn-nAbs levels of 66 PD patients and 69 healthy controls were assessed using a validated ELISA assay. Moreover, potential sources of error variance including unspecific ELISA background signals, free serum hemoglobin concentrations, α-Syn plate coating procedures, and differences in α-Syn-nAbs standards, were investigated. PD patients and controls did not differ in serum (p = .49) nor cerebrospinal fluid (p = .29) α-Syn-nAbs levels. Interestingly, free serum hemoglobin concentrations were negatively correlated with α-Syn-nAbs levels in controls (Spearman = −.41, p<.001), but not in PD patients ( = .16, p = .21). ELISA α-Syn plate coating procedures impacted inter-assay variability (same day coating: 8–16%; coating on different days: 16–58%). α-Syn-nAbs standards from different purification batches differed regarding optical density measured in ELISAs suggesting differences in α-Syn affinity. While α-Syn-nAbs levels may represent a potential PD biomarker, several methodological issues have to be considered to increase reproducibility of α-Syn-nAbs findings. Further studies using standardized protocols minimizing sources of error variance may be necessary to establish a reliable PD α-Syn-nAbs biomarker.

OS089. Elevated levels of the heme scavenger alpha-1-microglobulin in maternal plasma at the end of first trimester in patients who subsequently develop preeclampsia

Resent research has revealed an increased concentration of free fetal hemoglobin (HbF) in maternal serum from patients who subsequently develops preeclampsia (PE). In a previous study of 96 patients we have shown that HbF in combination with the heme-scavenger alpha-1-microglobulin (A1M) are potential predictive biomarkers of PE. In this validating case-control study we aimed to confirm the previous findings, that A1M is elevated in the maternal circulation at the end of first trimester in patients who subsequently develops PE. In this study A1M was measured in plasma instead of serum. Patients were recruited from an ongoing prospective study of new biomarkers to predict and diagnose PE. In total we included 84 patients. 8 patients subsequently developed PE, 4 developed pregnancy induced hypertension (PIH) and 72 were controls with uncomplicated pregnancies. The plasma samples were all taken at 7+0-18+0 weeks of gestation (mean 12+1) and analyzed for concentrations of A1M with Radioimmuno Assay (RIA). This method has been previously described in details. Statistics was performed using one-way ANOVA. The mean plasma concentration of A1M in the PE group was 8.6mg/ml, 6.0 in the PIH group and 7.1mg/ml in the controls group. The PE group differed significantly from the controls group (p=0.004), whereas the PIH group did not differ significantly from the controls. Our findings in plasma confirm previous findings described for serum, i.e. A1M is significantly increased in in first trimester maternal plasma in patients who subsequently develops PE. Since A1M is the most efficient heme scavenger we suggest that A1M may be a physiological defense mechanism against the elevated levels of free HbF found in patients who subsequently develops PE or in patients with manifest PE. Furthermore, A1M did not increase in patients who develops PIH later in their pregnancies indicating its specificity for PE.

Potassium report of hemolyzed serum samples

The recommendations for unsuitable samples recently published in Clinical Chemistry and Laboratory Medicine by Lippi et al. (1) on behalf of the SIBioCSIMeL-CISMEL Italian Study Group on Extra-Analytical Variability is an important step for standardization in laboratory medicine. However, while I agree with most statements made by the authors, I believe that they should have also dealt with a relevant issue: appropriate action in the detection of hemolyzed samples. In their study, the authors suggest that all results from tests potentially affected by interfering free hemoglobin in serum might be suppressed. A study carried out by our team (2) demonstrated that hemolyzed samples are usually the consequence of incorrect procedures for blood drawing and conservation; however, a small, but significant, number of cases (3.2% of all hemolyzed samples) were due to in vivo hemolysis. In these cases, the correct and immediate reporting of the potassium concentration is required for the appropriate treatment of patients. Based on this observation we introduced a specific procedure in our laboratory in order to identify, report and point out the problem to the requesting physician. In particular, in cases of moderate hemolysis, potassium is referred with the comment: ‘‘possible overestimation of potassium concentration: exclude in vivo hemolysis or repeat sampling’’. This procedure allowed us to identify several cases of acute hemolysis, in particular in overt disseminated intravascular coagulation or acute poisoning. Moreover, we detected some cases of redcolored serum, caused by myoglobin in the crash syndrome and acute vascular occlusion. Finally, a clinical report published in the British Medical Journal supported our approach (3). To my knowledge no studies are available to demonstrate that the patient outcome is better if results are suppressed or referred with a comment, but the latter choice appears a more reliable measure in making an early diagnosis and providing appropriate treatment, in particular in the setting of critical and emergency care.

The elevated serum urea : creatinine ratio in canine babesiosis in South Africa is not of renal origin

Pigmented serum, usually due to free haemoglobin and/or bilirubin, is a common finding in dogs with babesiosis, resulting in interference with all biochemical tests that rely on photochemistry. This is particularly true of urea and creatinine determinations, complicating the diagnosis of acute renal failure, which is a serious complication of babesiosis. A disproportionately raised serum urea concentration of unknown origin occurs in severely anaemic canine babesiosis patients and gives rise to an increased serum urea:creatinine ratio. The assay for cystatin-C, an excellent measure of glomerular filtration rate, is unaffected by free serum haemoglobin, and due to its different intrinsic origins, is free of influence by the metabolic derangements and organ pathology, other than renal disease, encountered in canine babesiosis. Serum cystatin-C was used to compare the concentrations of serum urea and serum creatinine in dogs with the severely anaemic form of canine babesiosis as well as a canine babesiosis-free reference group. Mean serum urea and mean serum urea:creatinine ratio were significantly elevated in the babesia-infected group relative to the reference population in this study. Mean serum creatinine and mean serum cystatin-C were within the reference ranges. Therefore an elevated urea:creatinine ratio in canine babesiosis in the presence of a normal serum creatinine concentration is considered to be caused by an elevated serum urea concentration and is most likely of non-renal origin. Serum creatinine was therefore as specific a measure of renal function as serum cystatin-C in canine babesiosis in this study. The sensitivity of serum creatinine as a measure of renal function was not established by this study. Serum urea, however, proved to be of little use compared to serum cystatin-C and serum creatinine. Serum urea should therefore not be used to diagnose renal failure in canine babesiosis.

Nitric oxide metabolites in naturally occurring canine babesiosis

Babesiosis, caused by the virulent haemoprotozoan parasite Babesia canis rossi, is an important disease of dogs in South Africa. The nitric oxide metabolites, nitrate and nitrite (collectively termed reactive nitrogen intermediates or RNIs) were measured in admission sera from dogs in a babesiosis-endemic area. Five groups were prospectively studied: mild uncomplicated ( n=9), severe uncomplicated (severe anaemia) ( n=10) and complicated babesiosis ( n=11); and two groups of healthy aparasitaemic dogs: endemic controls from the study area ( n=10) and experimental dogs kept in tick-free conditions ( n=10). Four measures of RNI production were studied: (i) serum RNI; (ii) serum RNI/creatinine ratio; (iii) fractional clearance of RNI (FC RNI); (iv) fractional excretion of RNI (FE RNI). Marked elevations of serum RNI occurred in only two dogs, both in the severe uncomplicated group. The highest concentration (log value 5.29 μmol/l) was in a dog that died, but concentrations in the other four dogs that died were unremarkable (0, 0.34, 1.66 and 2.64 μmol/l). Age, appetite and free serum haemoglobin were significant covariates for measures of RNI production. There were no significant differences among the babesiosis groups for serum RNI. Adjustment for creatinine had minor effects on the results. All babesiosis groups had significantly higher serum RNI and RNI/creatinine than the tick-free control group, but did not differ from the endemic controls except for the severe uncomplicated group, which had higher RNI/creatinine. The complicated group had significantly lower FC RNI and FE RNI than all other groups, except for the tick-free control group, which had similar FE RNI. The results indicate that, in an endemic area, measures of RNI production are unlikely to be useful indicators of severity or outcome in canine babesiosis.

Percutaneous fragmentation of pulmonary emboli in dogs with the impeller-basket catheter.

A new catheter system for rapid percutaneous fragmentation of pulmonary emboli was tested in nine dogs. The system consisted of a high-speed rotating impeller, placed within the center of a self-expandable basket. Preformed radiopaque emboli were introduced via the right external jugular vein. In nine tests in five dogs, a single embolus was launched after the right pulmonary artery had been balloon occluded and the impeller-basket catheter had been positioned into the left pulmonary artery. Seven of nine emboli were completely fragmented, each within less than 10 s. In two tests, performed in two different dogs, the catheter was positioned after complete embolization of the left pulmonary artery. In both cases, the central pulmonary arteries could be fully, and the segmental arteries partly, recanalized. Mobility of the system within the pulmonary arteries was limited. There was no evidence of wall damage at the rotation site of the impeller. Free serum hemoglobin did not increase after treatment. We conclude that the device can be safely operated in the left pulmonary artery system of dogs. It causes no significant hemolysis, and is able to accomplish rapid recanalization of the central arteries. Due to limited steerability, occluded side branches cannot be treated consistently.

Red blood cell T-activation and hemolysis in surgical intensive care patients with severe infections.

The exposure of Thomsen-Friedenreich (T) antigens on RBCs, serum neuraminidase, and serum hemoglobin levels were investigated in 53 adult surgical intensive care unit (ICU) patients with septicemia. Unmasked T-antigens were assayed by a hemagglutination test using peanut agglutinin (PNA) (direct anti-T test), and by an indirect anti-T test employing rabbit anti-PNA globulin. RBC T-activation was demonstrated in 17/53 patients (32%); in 2/53 patients (4%) the direct anti-T test was positive, indicating strong T-exposure. No polyagglutination phenomena were observed. Serum neuraminidase was elevated in 12/17 (71%) patients with T-activation and in 7/36 (19%) patients without T-activation. Free serum hemoglobin was elevated in 12/17 (71%) patients with T-activation and in 5/36 (14%) patients without T-activation. Correlations between T-activation and serum neuraminidase and between T-activation and serum hemoglobin were significant (p less than 0.001). Potentially neuraminidase-releasing bacteria were demonstrated in 13/17 (76%) patients with RBC T-exposure. We conclude that neuraminidase-induced RBC T-activation and subsequent hemolysis may be involved in the pathomechanism of hemolytic anemia in patients with severe infections.

Free Serum Hemoglobin in Syringe-Drawn and Vacuum Tube-Drawn Specimens

The amount of free serum hemoglobin produced secondary to in vitro hemolysis during the performance of a venipuncture was evaluated. Blood specimens were drawn using both syringe and vacuum tube apparatus for collection. All specimens were subjected to the same clotting, centrifugation, and separation procedures, and free serum hemoglobin was determined by the benzidine spectrophotometric method. The mean serum hemoglobin concentration of 1.41 mg dL for the syringe-drawn specimens slightly exceeded the normal mean of 1.3 mg dL, while the mean of 8.77 mg/dL for the vacuum tube-drawn specimens significantly exceeded the normal mean. The mean of the vacuum tube specimens represented a significant difference from the mean concentration of the syringe-drawn specimens, as determined by Student's t test at the P ≤ 0.001 level. Although hemolysis occurs to a significant extent with the vacuum tube system, the hemoglobin content of such specimens will not interfere with most clinical laboratory tests.

Effects of ethanol infusion on serum hemoglobin and bile pigments

Efforts to explain the apparent association between alcoholic liver disease and pigment gallstones have, to date, resulted in conflicting conclusions. While some clinical studies suggest hemolysis as the major pathogenetic factor other human and laboratory investigations describe increases in biliary pigment occurring in the absence of detectable hemolysis. In the present acute experiments various ethanol solutions ranging from 25 to 50% (v/v) were infused into miniswine to determine their effects on free hemoglobin in serum and total and unconjugated bilirubin in bile. Rapid and parallel increases in serum ethanol and serum hemoglobin levels were noted. Gradual and reversible increases in unconjugated and total bilirubin were observed after 1 hr. For unconjugated bilirubin the peak levels ranged from two times baseline levels at 25% (v/v) to 12 times baseline levels at 50%. When maximum values of hemoglobin and unconjugated bilirubin were compared in all experiments a strong positive correlation was confirmed. These findings suggest that the acute intravenous administration of ethanol causes intravascular hemolysis over a wide range of concentrations and results in the secretion of significant levels of bilirubin into bile. The role of this mechanism in pigment cholelithiasis remains to be determined.

拡大血液希釈性自己輸血に関する研究

Surgical hemorrhage (1413±1245ml), which was caused during radical operation of uterine cancer in 9 paitents, was treated with perioperative autologous donation (1200ml) supported by concomittant infusion of dextran solution and intraoperative blood salvage using Hemonetics Cell Saver®.By the intraoperative blood salvage, 255±134ml of suspension of washed red cells was obtained.Approximately 400ml of the autodonated blood was infused to maintain the circulating blood volume until the surgical blood loss reached to 800ml.In the meantime, the bled blood in operation field was salvaged and filtered by Bentley Reservoir Q-220F®. The red cells were washed and concentrated to 42.1±17.4% of Hematocrit by Cell Saver. And then infused through a transfusion set mounted Paul's microfilter.After completion of the transfusion, the circulating red cell mass remained in 71.1% of the initial value and the platelet count in 75.8%. The free hemoglobin in serum decreased from 31.5mg/dl to 22.9 after the transfusion and remained in the similar level for the subsequent seven days.No bacterial contamination was detected in the washed and packed red cells.