Decrease In Haptoglobin Research Articles

Characterization and clinical significance of hemolysis after pulsed-field ablation for atrial fibrillation: insights from a multicentric analysis

Abstract Background Pulsed-field ablation is a novel non-thermal ablation modality which is being increasingly used in clinical practice for the treatment of atrial fibrillation (AF). While the susceptibility of erythrocytes to electroporation is well-established, the effect of cardiac PFA technologies on erythrocyte destruction has not been described so far. Purpose The aim of this study was to investigate whether PFA induces clinically significant hemolysis. Methods We included 187 patients undergoing AF catheter ablation with either PFA (n=145) or RFA (n=42) at four high-volume centers. PFA was performed using a pentaspline catheter (biphasic, bipolar pulses of 2kV) and RFA with a 3.5 mm irrigated-tip catheter (40-60 W). The lesion set comprised pulmonary vein isolation (PVI) for paroxysmal AF and PVI ± additional lesions/lines for persistent AF. Established biomarkers of hemolysis, anemia and renal function were analyzed in blood samples obtained at baseline (T1), at the end of ablation (T2) and 24 hours after the procedure (T3). Results Baseline characteristics were well-balanced between groups (65.5 ± 9.6 years, 72.7% male, p>0.05). The ablation procedures comprised a mean of 61.4 ± 27.2 deliveries (PFA group) and a mean RF duration of 31.0 ± 16.3 min (RFA group). Atrial ablation in addition to PVI was performed in 55.2% vs. 61.9% patients, respectively (p=0.469). The direct hemolysis marker free plasma hemoglobin was significantly higher with PFA (592.8 ± 330.6 mg/dl) than with RFA (186.6 ± 215.8 mg/dl, p<0.001) at the end of ablation. A strong correlation was found between free plasma hemoglobin and PFA deliveries (Pearson r=0.615, p<0.001). PFA was further associated with a 2.2-fold increase in bilirubin (21.3 ± 11.3 vs. 9.6 ± 4.7 µmol/l), a 2.4-fold decrease in haptoglobin (0.5 ± 0.4 vs. 1.2 ± 0.4 g/l) and a 1.8-fold increase in LDH (352.7 ± 115.7 vs. 192.2 ± 51.6 U/l, T3 vs. T1, all p<0.001). There were no significant changes in bilirubin and haptoglobin in the RFA group (p>0.05). A slight decrease in red blood cells, hemoglobin and hematocrit was detected after both PFA and RFA (Abstract Figure), with no significant differences between groups (all p>0.05). While mean creatinine levels and glomerular filtration rates did not significantly change after ablation in either group, acute kidney injury occurred in 4/124 (3.2%) with PFA vs. 0/32 (0%) with RFA (p=0.303). Conclusion Intravascular hemolysis is a frequent finding after PFA for AF and increases with the number of PFA deliveries. While PFA was not associated with clinically significant anemia, the incidence of acute kidney injury in the present study warrants future large-scale investigations assessing the biological impact and nephrotoxicity of PFA-induced hemolysis.Abstract Figure

Effects of milk replacer allowances and levels of starch in pelleted starter on nutrient digestibility, whole gastrointestinal tract fermentation, and pH around weaning

The objectives of this study were to examine the effects of pelleted starter diets differing in starch and neutral detergent fiber (NDF) content when fed differing levels of milk replacer (MR) on nutrient digestibility, whole gastrointestinal tract fermentation, pH, and inflammatory markers in dairy calves around weaning. Calves were randomly assigned to 1 of 4 dietary treatments (n = 12 per treatment) in a 2 × 2 factorial design based on daily MR allowance and amount of starch in pelleted starter (SPS): 0.691 kg of MR per day [dry matter (DM) basis] with starter containing low or high starch (12.0% and 35.6% starch on DM basis, respectively), and 1.382 kg of MR per day (DM) with starter containing low or high starch. All calves were housed in individual pens with straw bedding until wk 5 when bedding was covered. Calves were fed MR twice daily (0700 and 1700 h) containing 24.5% crude protein (DM) and 19.8% fat (DM), and had access to pelleted starter (increased by 50 g/d if there were no refusals before weaning and then 200 g/d during and after weaning) and water starting on d 1. Calves arrived between 1 and 3 d of age and were enrolled into an 8-wk study, with calves undergoing step-down weaning during wk 7. Starting on d 35, an indwelling pH logger was inserted orally to monitor rumen pH until calves were dissected at the end of the study in wk 8. Higher SPS calves showed an increase in rumen pH magnitude (1.46 ± 0.07) compared with low SPS calves (1.16 ± 0.07), a decrease in rumen pH in wk 8 (high SPS: 5.37 ± 0.12; low SPS: 5.57 ± 0.12), and a decrease in haptoglobin in wk 8 (high SPS: 0.24 ± 0.06 g/L; low SPS: 0.49 ± 0.06 g/L). The majority of differences came from increased starter intake in general, which suggests that with completely pelleted starters the differences in starch and NDF do not elicit drastic changes in fermentation, subsequent end products, and any resulting inflammation in calves around weaning.

Anemias hemolíticas adquiridas y congénitas

Hemolytic anemias are a heterogeneous group of diseases with a common factor of a decrease in red blood cell survival (hemolysis). In this pathology, findings include an increase in reticulocytes, indirect bilirubin, and lactate dehydrogenase (LDH) and a decrease in haptoglobin. The intensity of clinical symptoms will be more or less severe based on the degree of hemolysis. Hemolytic anemias can be classified in two big groups: congenital and acquired hemolytic anemias. It is important to establish a good etiological diagnosis in order to be able to differentiate between them and thus establish the corresponding treatment, given that each requires specific management.

The utility of a monocyte monolayer assay in the assessment of intravenous immunoglobulin–associated hemolysis

Hemolysis following the administration of intravenous immunoglobulin (IVIG) is an important adverse event (AE). While the monocyte monolayer assay (MMA) has been used to predict in vivo hemolysis when serologically incompatible blood may be transfused, it has also been shown to correlate with IVIG-associated hemolysis. In this study, the MMA was examined for its utility in assessing the risk of hemolysis after IVIG. Forty-two non-blood group O patients receiving high-dose IVIG (≥2 g/kg) were examined using an autologous and allogeneic MMA. Hemolysis was defined by a drop in hemoglobin of ≥1 g/L, a positive direct antiglobulin test (DAT) and eluate, and a decrease in haptoglobin or increase in lactate dehydrogenase and/or reticulocytes. Forty-two patients provided 50 assessable postinfusion samples, with hemolysis observed in 20 (40%) of cases. Autologous MMA using post-IVIG red blood cells significantly correlated with clinical outcomes when compared to allogeneic MMA (P = .0320 vs .5806, t test). No significant difference in receiver operating characteristics was observed when comparing autologous MMA testing against DAT for the diagnosis of IVIG-associated hemolysis. However, when using samples collected 5 to 10 days after receipt of high-dose IVIG, the autologous MMA had higher sensitivity than the DAT. MMA testing with autologous monocytes collected 5 to 10 days after receipt of high-dose IVIG can be used for the diagnosis of IVIG-associated hemolysis and may be of particular value in cases in which the Day 5 to 10 DAT is negative.

Акушерский атипичный гемолитико- уремический синдром: виновата беременность или ее осложнения?

Atypical hemolytic-uremic syndrome (aHUS) is one of the most severe forms of thrombotic microangiopathy (TMA) that might develop during pregnancy and after childbirth. It is conditioned by a severe, progressive course of disease, unfavourable prognosis and difficult differential diagnosis between this and other forms of TMA – first of all, with classical obstetric complications: preeclampsia (PE) and HELLP syndrome. It is considered that pregnancy-associated aHUS (P-aHUS) is triggered by pregnancy itself. But an analysis of publications dealing with pregnancy-associated aHUS and our own experience accumulated by the present time indicate that the overwhelming majority of P-aHUS cases develop not during pregnancy, but after delivery, and almost always an acute TMA episode is preceded by various complications of pregnancy (PE, bleeding, placental abruption, antenatal intrauterine fetal death), infections, operative interventions, etc. As is known, they all might be regarded as a complement-activating conditions (CACs). The objective. To study the effects of obstetric complications, mainly preeclampsia, on the development, course and prognosis of pregnancy-associated aHUS. Patients and methods. From 2011 to 2019, 69 patients aged 16–44 years were examined, in whom aHUS developed during pregnancy or directly after childbirth. Most women (47 of 69, 68%) were secondiparas without any bad obstetric history. In 62 (90%) of 69 patients disease developed during the postpartum period at terms from several hours to 8 days after childbirth. Results. All patients had a full symptom complex of TMA: microangiopathic hemolytic anaemia (lower haemoglobin levels 62.3 ± 15.0 g/l, higher lactate dehydrogenase levels 2683.7 ± 2143.1 U/l, schisocytosis, a decrease in haptoglobin), thrombocytopenia (50.0 ± 32.8 thous. per μl), involvement of the kidneys (higher creatinine levels 509.0 ± 349.8 μmol/l, oligoanuria) and other organs (liver, heart, central nervous system, lungs). Severe multiple organ failure was observed in 91.3% of patients. In all cases, the development of aHUS was preceded by additional CACs, the most common of which were caesarean section (73.5%), bleeding (69.5%) and PE (69.5%), although the number of CACs did not have a significant effect on the severity and outcome of aHUS. Neither were found significant differences in the clinical and laboratory manifestations in patients with the presence/absence of caesarean section, PE or bleeding (each taken separately). 40 (58%) of 69 patients received a complement-blocking therapy with Eculizumab. In patients receiving Eculizumab, compared to the patients who received only fresh frozen plasma therapy (29 women), complete recovery of renal function was noted in 26 (65%) vs 10 (34.5%) women, 5 (12.5%) vs 5 (17.5%) remained dialysis-dependent, 5 (12.5%) vs 11 (38%) died. Nineteen (47.5%) of 40 patients received only a full course of induction therapy or only 1–2 infusions of Eculizumab, in three more patients the drug was discontinued after 4–24 months. None of the women had a recurrence of TMA after discontinuation of Eculizumab. Conclusion. In all women with P-aHUS pregnancy was combined with additional CACs, as a rule, their number was >3. In 69.5% of cases, the disease was preceded by PE, which, in its turn, is considered as a specific obstetric variant of TMA. Considering interrelationships between PE and P-aHUS, we can suppose that glomerular capillary endotheliosis, characteristic for PE and conditioned by an imbalance between PlGF- and sFlt1-factors of angiogenesis/antiangiogenesis, promotes additional activation of the complement, creating preconditions for fast generalisation of endothelial lesions in patients with genetic defects in the complement system, which gives every reason to discuss the possibility of transformation of PE into aHUS. In this connection, all patients with PE, especially with severe one, should be referred to a group of risk for possible postpartum generalisation of the microangiopathic syndrome. It would be appropriate to regard P-aHUS as a heterogeneous group that includes both «classical» aHUS, and «secondary» HUS, not associated with constitutional ysregulation of the complement system. Early beginning of Eculizumab therapy permits not only to save the life of a patient with aHUS, but also to fully recover their health. In case of development of «secondary» aHUS, a course of Eculizumab might be reduced to 1–2 infusions. Key words: pregnancy-associated atypical haemolytic-uraemic syndrome, pregnancy, thrombotic microangiopathy, Eculizumab

Inferior Vena Cava Filters as a Potential Non-immune Cause of Low Serum Haptoglobin

Objective: Serum haptoglobin level is commonly used as a marker of hemolysis. We hypothesized that inferior vena cava (IVC) filters may be a potential non-immune etiology for subclinical hemolysis and low haptoglobin in some patients, after encountering a patient with an IVC filter, mild macrocytic anemia and unexplained severe haptoglobinemia. IVC filters can be associated with pressure gradients and turbulent blood flow in the presence of trapped clots and thrombus formation. Methods: Prospective study in patients undergoing IVC filter placement at our institution over a 3-month period in 2008. All patients had a haptoglobin level prior to filter placement, and then daily for a period of 3-14 days. The absolute and relative change in haptoglobin compared to pre-procedure levels was determined. Results were evaluated by t-test (paired, 2-tail). Statistics and graphics were performed with commercial software. Results: A total of 22 patients underwent IVC filter placement. Four patients were excluded due to low preprocedure haptoglobin levels. At least one post-procedure haptoglobin values was available until day 3 in 18/18 eligible patients. Nine patients were followed for a period of 1-2 weeks. Although the majority of patients had an increase in haptoglobin after IVC filter placement, 3/9 (30%) had >50% decrease in haptoglobin by day 7, including one patient with severe haptoglobinemia by day 14. Patients with decreased haptoglobin levels also had decreases in platelet count over the same period. Conclusion: These results suggest that IVC filters may be another non-immune, device-related cause of decreased haptoglobin in some patients.

Evaluation of hemolysis in microcatheter directed blood infusion at different flow rates for transarterial salvage reperfusion: In-vitro study.

Microcatheter directed blood reperfusion is an endovascular salvage option for acute cerebral artery occlusions. It has not been investigated whether this technique may be associated with hemolysis. Analysis of hemolysis during blood infusion through different microcatheters and infusion rates to assess related risks. Four microcatheters with different inner diameters were perfused with blood samples at three infusion rates. Hemolytic markers including lactate-dehydrogenase (LDH) and haptoglobin were analyzed. Samples before and after blood infusion were compared using Student's t-test. Flow-related degree of hemolysis was analyzed with regression analysis. Resulting shear stress was calculated and correlated with LDH and haptoglobin. Significant increase of LDH and decrease of haptoglobin was found after blood reperfusion through small microcatheters at progressive flow rates (p<0.05). No hemolysis was found with larger diameter microcatheters at all flow rates (p>0.05). Correlation between shear stress, LDH and haptoglobin was r=0.86 and r=0.75, respectively. Progressive hemolysis occurs during blood perfusion of small lumen microcatheters at increasing flow rates. This phenomenon may be related to turbulent flow, exposure time and increased shear stress. Larger microcatheters did not induce hemolysis and may be the preferred choice for stroke reperfusion.

Natural cocoa consumption: Potential to reduce atherogenic factors?

Short-term consumption of flavanol-rich cocoa has been demonstrated to improve various facets of vascular health. The purpose of the present study was to determine the effect of 4 weeks of natural cocoa consumption on selected cardiovascular disease (CVD) biomarkers in young (19–35 years) women of differing body mass indices (BMI; normal, overweight or obese). Subjects (n=24) consumed a natural cocoa-containing product (12.7 g natural cocoa, 148 kcal/serving) or an isocaloric cocoa-free placebo daily for 4 weeks in a random, double-blind manner with a 2-week washout period between treatment arms. Fasted (>8-h) blood samples were collected before and after each 4-week period. Serum was analyzed to determine lipid profile (chemistry analyzer) and CVD biomarkers (26 biomarkers). EDTA-treated blood was used to assess monocytes (CD14, CD16, v11b and CD62L), while citrate-treated blood was used to measure changes in endothelial microparticles (EMPs; CD42a−/45−/144+) by flow cytometry. Natural cocoa consumption resulted in a significant decrease in haptoglobin (P=.034), EMP concentration (P=.017) and monocyte CD62L (P=.047) in obese compared to overweight and normal-weight subjects. Natural cocoa consumption regardless of BMI group was associated with an 18% increase in high-density lipoprotein (P=.020) and a 60% decrease in EMPs (P=.047). Also, obese subjects experienced a 21% decrease in haptoglobin (P=.034) and a 24% decrease in monocyte CD62L expression in (P=.047) following 4 weeks of natural cocoa consumption. Collectively, these findings indicate that acute natural cocoa consumption was associated with decreased obesity-related disease risk. More research is needed to assess the stability of the observed short-term changes.

HBV and HCV coinfection associated with warm-type autoimmune hemolytic anemia: a case report.

Received/Gelis tarihi : December 15, 2012 Accepted/Kabul tarihi : April 30, 2013 To the Editor, The hepatitis virus may play an important role in autoimmune hemolytic anemia (AIHA). We herein report the case of a patient presented to our hospital with hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfection who developed warm-type AIHA. The anemia improved, followed by declined viral levels, after rituximab plus antiviral therapy. To the best of our knowledge, this is the first reported case of HBV and HCV coinfection associated with warm-type AIHA. Written informed consent was obtained from the patient. A 66-year-old woman presented to the department of internal medicine of our hospital on 31 August 2010 with jaundice, inappetence, dizziness, palpitation, and fatigue. A month before admission, the patient complained of fatigue and palpitation, particularly aggravated after exercise, but she did not pay attention to it due to relief of these symptoms at rest. Three weeks later, the patient felt inappetence and dizziness, and her fatigue and palpitation did not go into remission at rest. Her daughter noticed her jaundice at this point and directed her to our hospital. She did not have past medical history of diffuse connective disease, lymphoproliferative disease, transfusion, favism, or allergic contact dermatitis. She had no related social history of cigarette, alcohol, or drug use. Her general physical examination revealed icteric skin and sclerae. Her abdominal examination showed a slightly increased spleen located in the left-upper abdomen. Complete blood count showed severe decrease of red blood cell count (1.39x1012/L), hemoglobin (53 g/L), and platelet count (73x109/L), in addition to significantly increased reticulocyte percentage (19%). Blood biochemistry revealed elevated total bilirubin (6.62 mg/dL), unconjugated bilirubin (4.95 mg/dL), and lactic dehydrogenase (528 U/L), with remarkable decrease of haptoglobin (0.3 g/L). The immunological test displayed

Cardiopulmonary Bypass Is Associated With Hemolysis and Acute Kidney Injury in Neonates, Infants, and Children*

This pilot study assesses the degree of hemolysis induced by cardiopulmonary bypass and determines its association with acute kidney injury in pediatric patients. Further, it evaluates the degree to which the use of urinary biomarkers neutrophil gelatinase-associated lipocalin and cystatin C correlate with the presence of acute kidney injury and hemolysis following cardiopulmonary bypass. Prospective observational study. A 13-bed pediatric cardiac ICU in a university hospital. Children undergoing cardiac surgery with the use of cardiopulmonary bypass. None. Blood and urine samples were obtained at multiple time points before and after cardiopulmonary bypass. Hemolysis was assessed by measuring levels of plasma hemoglobin and haptoglobin. Acute kidney injury was defined as a doubling in serum creatinine from preoperative baseline and by using the pediatric-modified RIFLE criteria. Urinary neutrophil gelatinase-associated lipocalin and cystatin C levels were measured. A total of 40 patients (range, 3 d to 4.8 yr) were enrolled. Plasma hemoglobin levels increased markedly on separation from cardiopulmonary bypass with a concurrent decrease in haptoglobin. This was associated with an increase in protein oxidation in the plasma. Hemolysis was more evident in younger patients with a longer duration of bypass and in those requiring a blood-primed circuit. Forty percent of patients had a doubling in serum creatinine and acute kidney injury was developed in 88% of patients when defined by the pediatric-modified RIFLE criteria. Controlling for cardiopulmonary bypass time, persistently elevated levels of plasma hemoglobin were associated with a five-fold increase in acute kidney injury. Further, urinary neutrophil gelatinase-associated lipocalin measured 2 hours after separation from cardiopulmonary bypass was associated with acute kidney injury and with elevations in plasma hemoglobin. Cardiopulmonary bypass in pediatric patients results in significant hemolysis, which is associated with the development of acute kidney injury. The biomarker neutrophil gelatinase-associated lipocalin correlates with both acute kidney injury and hemolysis in this population.

Evaluation of a New Method for Measuring Vascular Access Recirculation

Together with Nikkiso in Shizuoka, Japan, we developed a new method for measuring the rate of vascular access recirculation by the blood volume monitor. This measurement is performed via a method of dilution that employs a marker produced by rapid ultrafiltration using a dialysis machine. In this paper, we evaluate the reliability and safety of this machine, in vitro and in vivo. The safety of this method was evaluated by investigating hemolysis after rapid ultrafiltration. The measurement of free hemoglobin, potassium and haptoglobin in the circulating blood were performed before and after rapid ultrafiltration. No data was found to indicate hemolysis in vivo, detected by an increase in potassium or a decrease in haptoglobin. Evaluation of reliability in an experimental system was also performed on an in vitro recirculation system at a rate of 0, 10, 25, 50, and 70%. Almost all of the measured rates were within ± 10% of the theoretical rate. We performed 20 hemodialysis experiments with vascular access recirculation applying this monitor and simultaneous urea and creatinine dilution methods, which were the recommended standard measurements for vascular recirculation. In 53 measurements of standard vascular shunts with no postural change, differences of the results between the monitor and both dilution methods were only 4.0% and 3.2%, respectively. Regression analysis showed a significant and positive correlation between them (P < 0.0001). We conclude that this new method for measuring vascular access recirculation is applicable in terms of both accuracy and ease of operation.

Serum acute phase proteins concentrations in dogs during experimentally short-term induced overweight. A preliminary study

The purpose of the study was to analyse serum concentrations of four different positive acute phase proteins (APPs): C-reactive protein, haptoglobin, serum amyloid A and ceruloplasmin in a model of experimentally short-term developed obesity in Beagle dogs. All APPs were quantified by commercially available ELISA methods and C-reactive protein was also determined by a highly sensitive time-resolved fluorometric assay. There were no significant differences between APPs concentrations at the beginning and the end of the study in groups of dogs that increased their body condition scores. In addition, dogs with body condition scores of 4 and 5 did not shown significant differences for any of the acute phase proteins studied compared with control dogs of BCS of 3, with exception of a decrease in haptoglobin. It was concluded that overweight induced in the experimental conditions of this study does not produce a significant change in acute phase proteins.

Extracellular haemoglobin, oxidative stress and quality of red blood cells relative to perioperative blood salvage

The quality of blood-pack units in perioperative blood salvage was studied with respect to the presence of extracellular haemoglobin (Hb) and in terms of oxidative stress and the mechanical properties of red blood cells (RBC). The study was performed on blood-pack units and patients' blood after retransfusion. Results were compared to those obtained in patients prior to autotransfusion. Free Hb, non-protein thiols (as antioxidant marker) and markers of oxidative stress (conjugated dienes, thiobarbituric reactive substances and protein carbonyl content) were measured. The mechanical properties of RBC were evaluated by their osmotic fragility, membrane fluidity by measuring the fluorescence anisotropy of an extrinsic probe and permeability by measurement of extracellular K(+) and lactate dehydrogenase. Despite washing, extracellular Hb concentrations remained high (up to 0.7 g/L in blood-pack units) and was associated with a decrease of haptoglobin in patients, despite a concomitant inflammatory syndrome. In blood-pack units, we observed a decrease in antioxidant markers along with an increase in oxidative stress markers in association with an alteration of RBC membrane properties. Haemolysis must be limited during perioperative blood salvage in order to prevent exposure to oxidative stress and improve the efficiency of autotransfusion.

Haptoglobin serum concentration is a suitable biomarker to assess the efficacy of a feed additive in pigs

Levels of haptoglobin and Pig-major acute phase protein (MAP) were analysed in animals from a commercial herd receiving or not a diet enriched with an additive. The group receiving the additive exhibited a decrease in haptoglobin after 3 weeks, suggesting that a better health status has been established, together with an improvement in total body weight and average daily gain. In contrast, Pig-MAP does not significantly change under these conditions. Aujeszky live modified vaccination, which is compulsory in Spain, did cause a significant increment in haptoglobin serum concentration although it did not affect Pig-MAP. The response of acute phase proteins to vaccination was similar in both control and additive-treated groups. Interleukins (IL)-1β and IL-6 was below the detection limits in most of the animals. In conclusion, this study shows that haptoglobin serum concentration, but not Pig-MAP, is a good biomarker to monitorize production parameters and for monitoring Aujeszky modified live vaccine in pigs reared under standard commercial conditions.

Safety and Long-Term Efficacy of Eculizumab in a Renal Transplant Patient with Recurrent Atypical Hemolytic–Uremic Syndrome

To the Editor: Atypical HUS develops as the result of unregulated complement progression and has been shown to cause microangiopathic hemolytic anemia. Plasmapheresis is currently used in an attempt to control the progression of the complement cascade, but this therapy is cumbersome and not effective in all patients. Eculizumab, a monoclonal antibody that targets complement factor C5, blocks activation of the terminal complement cascade and the generation of the proinflammatory and prothrombotic molecules C5a and C5b-9. Eculizumab has been approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) (1). In a recent study, a single dose of 600 mg of eculizumab appeared to resolve hemolysis and thrombocytopenia, and led to recovery of renal function in a transplant patient during an episode of aHUS (2). We report here on the efficacy of eculizumab in a patient who presented a recurrence of atypical hemolytic syndrome 3 years after renal transplantation. The patient is a previously described 42-year woman with familial aHUS and a heterozygous gain of function mutation (R570Q) in the C3 gene (3). She developed end-stage renal disease at age 30. Kidney transplantation was performed at age 34, and aHUS recurred 5 months later, leading to the loss of the graft after 2 years. She received a second transplantation with a recurrence of aHUS 3 years later, but recovered renal graft function under intensive plasma exchanges over 9 months (47 plasmaphereses). Four months later, the patient presented with septicemia and acute renal failure. Her haptoglobin became undetectable (<0.15 g/L), and schistocytes increased to 3.7% suggesting an acute thrombotic microangiopathic (TMA) exacerbation, which was confirmed by renal biopsy. The administration of plasma exchanges (19 treatments over 5 weeks) resulted in an improvement in the ongoing TMA. However, the patient continued to suffer from severe fatigue and daily episodes of diarrhea and chose to discontinue plasmapheresis because of its perceived negative impact on her quality of life. Eculizumab was proposed as an alternative therapy, the patient being fully informed of the lack of both registered clinical trials and evidence concerning its efficacy in aHUS patient. The recommended ‘meningococcal polysaccharide, diphtheria toxoid conjugate vaccine’ and patient consent form were provided before initiating eculizumab. Treatment with eculizumab was initiated 4 days after the final plasmapheresis. Based upon the dose regimen determined for PNH, the patient received four doses of eculizumab, 900 mg IV every 7 days, followed by a maintenance dose of 1200 mg every 14 days. Schistocyte count, hemolysis and renal function were monitored. After 7 months of eculizumab treatment, and without concomitant plasmapheresis, schistocytes decreased to 0.5%, haptoglobin increased to within normal limits, creatinine levels stabilized and no further episodes of diarrhea were reported. The need for blood transfusions was significantly reduced and they were stopped 4 months after continued eculizumab treatment. Interestingly, the ninth infusion of eculizumab was delayed by 6 days due to an episode of chest pain. This delay was temporarily associated with a return of hemolysis, deterioration of transplant function and the need for a final transfusion (Figure 1). We hypothesize that complement breakthrough due to a lapse in eculizumab dosing resulted in the return of TMA. In view of the stabilization of her aHUS, one may argue that eculizumab should be considered as permanent treatment for this particular patient. Response to chronic eculizumab therapy in a patient with atypical hemolytic–uremic syndrome. Eculizumab therapy was initiated in a renal transplant patient with aHUS that refused plasmatherapy. Renal function and hemolysis were monitored before and during treatment. Before eculizumab therapy, four blood transfusions were administered due to the ongoing hemolysis. After initiation of eculizumab therapy, hemolysis was controlled (as assessed by levels of schistocytes and haptoglobin). Blood transfusions were delivered due to metrorrhagia following contraception discontinuation and a lapse in eculizumab therapy. The patient has not required further transfusions for 85 days of treatment. A lapse in the eculizumab dosing regimen was temporally associated with an increase in schistocytes, a decrease in haptoglobin and a sharp rise in levels of creatinine indicating a return of TMA. Control of TMA was reestablished with the next dose of eculizumab and has continued to be controlled with ongoing treatment. In summary, these data suggest that chronic blockade of complement C5 with eculizumab in a patient with aHUS previously dependent on frequent plasmapheresis is safe, and leads to maintained renal function, reduced need for blood transfusions and control of TMA and hemolysis in the absence of plasmapheresis. It is obvious that a study aimed to confirm the efficacy of eculizumab in aHUS is needed. Valérie Chatelet, Véronique Frémeaux-Bacchi, Thierry Lobbedez, Maxence Ficheux and Bruno Hurault de Ligny declare no competing financial interests with Alexion Pharmaceuticals. Eculizumab was provided by Alexion Pharmaceuticals on an individual patient compassionate basis.

Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer

Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum samples from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum samples were processed either by linear (pH 4-7) or narrow range (pH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear pH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum samples collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.

New Alterations of Serum Glycoproteins in Alcoholic and Cirrhotic Patients Revealed by High Resolution Two-Dimensional Gel Electrophoresis

Plasma protein are synthesized and secreted by the liver. Several reports have shown that excessive consumption of ethanol interferes with the hepatic protein synthesis and/or secretion. This study was undertaken to identify the plasma/serum proteins altered in two groups of patients with different alcohol-related diseases: actively drinking alcoholic patients group without liver disease and alcohol cirrhotic patients group. Two-dimensional gel electrophoresis was used to separate proteins with high resolution. Proteins were detected by silver staining and glycoproteins were specifically visualized and analyzed after lectin blotting followed by chemiluminescence detection. Different protein alterations were identified in each group of patients. In the alcoholic group, two new glycosylation modifications of serum proteins were identified. An abnormal microheterogeneity of haptoglobin and α1-antitrypsin was detected in the serum of all alcoholic patients. We also characterized by two-dimensional gel electrophoresis the carbohydrate deficient transferrin. The modifications of haptoglobin, α1-antitrypsin and transferrin present a similar change of charge and molecular weight in the two-dimensional gel electrophoresis pattern. These qualitative estimations support the hypothesis of a general mechanism of liver glycosylation alteration of serum proteins induced by excessive alcohol consumption. The immunoglobulin alterations were easily visualized and identified for the cirrhotic and the alcoholic patients. And finally, the decrease of haptoglobin and albumin spots for cirrhotic patients was confirmed.

Hemolytic reactions mechanically induced by kinked hemodialysis lines

Ten hemolytic reactions occurred in our outpatient hemodialysis unit over a 12-month period (December 1989 to December 1990). Eight patients were hospitalized and one died. All patients developed severe abdominal or back pain an average of 2.5 hours into a 4-hour hemodialysis session using a bleach/formaldehyde reprocessed hollow-fiber cupraphan dialyzer (average reuse, three times) with blood flow rates of 375 mL/min. All had visible hemolysis in a spun hematocrit, seven had a significant decrease in hematocrit, and six developed pancreatitis. Hemolysis was further confirmed by a decrease in haptoglobin in all patients and an increase in lactic dehydrogenase in all but the last case. Investigation of each episode failed to find an abnormality in dialysate temperature or tonicity; dialysate or water levels of copper, zinc, nitrates, chloramine, or formaldehyde; or blood pump or venous alarm. Hemolytic reactions continued despite changing to 15-gauge needles, removing bleach from the reuse procedure, or stopping reuse. During the eighth episode, a kink was noted in the arterial blood line. Two subsequent hemolytic reactions occurred, and in each kinks were found in the arterial blood line, either in the excess tubing between the blood pump and drip chamber or in the predialyzer. No further hemolytic reactions occurred after changing to a new arterial blood line without redundant tubing and securing all lines. Hemolytic reactions occurring during hemodialysis have many etiologies, including mechanical trauma, which we report may result from kinking of dialyzer lines. With new blood lines on the market, attention to this aspect of dialysis is mandatory.

Efficacy of rhesus antibodies (Anti-Rh0 (D)) in autoimmune thrombocytopenia: correlation with response to high dose IgG and the degree of haemolysis.

We have compared the efficacy of high-dose IgG with that of Rhesus antibodies (anti-Rh0 (D)) in 5 patients with autoimmune thrombocytopenic purpura (3 adults and 2 children). Although only transient, high-dose IgG (0.4 g/kg X 5 days) was effective in all patients (peak values 50-200 X 10(9)/1), whereas anti-Rh0 (D) (11-20 micrograms/kg X 5 days) led to comparable results in only 3 patients (165 X 10(9)/1, 72 X 10(9)/1, 33 X 10(9)/1). This response to anti-Rh0 (D) was neither related to the degree of induced haemolysis (increase of LDH and decrease of haptoglobin) nor to the amount of IgG antibodies bound to red blood cells, as quantified by the 125-I-antiglobulin test. A decrease of platelet-associated IgG was recorded in 3 patients: 2 of them showed an improvement of platelet counts and in one of them there was no response. We conclude that the therapeutic response of high-dose IgG and anti-Rh0 (D) is independent of the degree of induced haemolysis and may not be predicted from the effectiveness of either therapy alone.

Treatment of autoimmune thrombocytopenic purpura with rhesus antibodies (anti-Rh0(D).

There is evidence that blockade of the reticuloendothelial system (RES) by sequestration of autologous red blood cells (RBC) leads to an elevation of platelet counts in immune thrombocytopenia. To substantiate this hypothesis, 10 Rh0(D)-positive adult patients (9 female, 1 male) with chronic autoimmune thrombocytopenic purpura (ITP) (1 to 21 years duration) were treated with low doses of intravenous IgG-anti-Rh0(D) (200 to 1,000 micrograms per dose; 300 to 3,600 micrograms per course; administration within 1 to 5 days). All patients improved clinically as indicated by cessation of bleeding. In eight out of ten patients there was a rise in platelet count. Platelet increments were excellent (greater than 100 X 10(9)/l) in one, good (50-100 X 10(9)/l) in three, fair (20-50 X 10(9)/1) in two and low (10-20 X 10(9)/1) in two patients. Splenectomized patients (N = 4) had a poorer response than non-splenectomized patients (N = 6) with mean increments of 16 X 10(9)/l (range 5-43 X 10(9)/l) versus 60 X 10(9)/l (range 10-110 X 10(9)/l). The increase in platelet counts persisted for seven to over 150 days. Transient and slight signs of haemolysis developed in seven out of ten patients (haemoglobin remained stable; increase of lactate dehydrogenase (greater than 250 IU/l) in four, decrease of haptoglobin (less than 60 mg/dl) in five patients). The direct antiglobulin test became positive in all cases due to IgG1 without complement fixation. We conclude that the interaction of antibody-coated RBC with macrophages (and, probably, other means of RBC alteration) is a feasible therapeutic approach in selected cases of ITP and related conditions.