Hemolytic Process Research Articles

Case Of Severe Unconjugated Hyperbilirubinemia In A Neonate Heterozygous For Gilbert Syndrome

We present an unusual case of unconjugated hyperbilirubinemia in a 6 day old infant. The bilirubin peaked at 20.1/ 0.6 mg/dL. A work-up for a hemolytic process or metabolic disorder was negative. Crigler-Najjar was suspected. Phenobarbital was given for 3 days with no significant change in bilirubin level. He was discharged home after 20 days of phototherapy with a bilirubin of 3.1/0.4 mg/dL. Five months later, his bilirubin was 0.2 mg/dL. His genetic testing came back positive for a heterozygous mutation for Gilbert Syndrome. In the UGT1A1 gene he had the following mutations: heterozygous *28 (TA 6/7) (c. 40-39insTA), heterozygous *60 (c-3275T>G), and heterozygous *93 (c.- 3152G>A). This result is consistent with a carrier state for unconjugated hyperbilirubinemia and may be associated with mild to moderate hyperbilirubinemia. The relevance of this haplotype of polymorphisms to hyperbilirubinemia in the neonate has not been established. Gilbert syndrome is the most common hereditary cause of increased bilirubin but is typically associated with mild hyperbilirubinemia, around 3 mg/dL. In the homozygous state, diminished bilirubin glucuronidation is observed but it is questionable if the same amount of decreased activity can be seen in the heterozygous state. It has been proposed that when additional mutations exist in conjunction with a heterozygous state, neonatal hyperbilirubinemia is more pronounced. We believe that unexplained unconjugated hyperbilirubinemia should raise the suspicion of a UGT1A1 gene mutation and should prompt genetic testing. Gilbert syndrome should be added to the differential of severe unconjugated hyperbilirubinemia in the neonate

Exploratory Study of Haptoglobin Levels by Genotype in Adult Sickle Cell Patients

Abstract 2145Patients with sickle cell disease (SCD) have both extravascular and intravascular hemolysis; it is estimated that 1/3 of the hemolytic process in SCD is intravascular. Hemoglobin (Hb) released from hemolyzed red blood cells is bound by haptoglobin (Hp) in a 1:1 stochiometric complex, which is then taken up by macrophages through the scavenger receptor, CD163. When the binding capacity of haptoglobin is exceeded, concentration of cell free Hb in plasma increases, excess cell free Hb is a nitric oxide (NO) scavenger and leads to a NO depletion state. This, in turn, leads to endothelial dysfunction with increased expression of adhesion molecules and inflammatory markers, platelet activation, and an inflammatory state. This process has been incriminated in the pathogenesis of certain complications of SCD and other chronic hemolytic states with a significant intravascular hemolytic component. These include increased pulmonary artery pressure (PASP), priapism, leg ulcers, and renal dysfunction. In addition to these deleterious effects of excess cell free Hb, the fate of Hb—Hp complexes may play a significant role in the pathogenesis of the inflammatory state. Hp is a polymorphic protein with two well characterized alleles, Hp-1 and Hp-2. A large body of evidence suggests that Hp-2 allele si a major susceptibility gene for the development of vascular complications (coronary artery restenosisand cardiovascular disease). This has been attributed to the generation of a more potent inflammatory response after the binding of the larger Hp-2/Hb complexes to the CD163 receptor. We previously reported on the distribution of Hp genotypes in our pediatric and adult populations [Hughes et al, Blood 106(11), 3805 30(b), December 2005] and more recently have shown that binding of Hp-2-Hb complexes to mononuclear cell cultures from SCD patients resulted in a 3–12 fold higher inflammatory cytokine release (GM-CSF, IL1, IL-10, and TNF) compared to that observed with Hb-Hp1 complexes [Bora et al, Blood 112(11):513(1441), 2008]. We studied the haptoglobin levels in the plasma of SCD patients on and off hydroxyurea (HU) and in relation to their Hp genotype. Since clinical assays for haptoglobin are rather insensitive below 6 mg/dl, we used a sensitive ELISA (Genway Biotech) technique to quantify Hp. Hp genotypes are determined by a previously published PCR procedure, which resulted in a 3245 bp product for Hp-1 and 4945 bp for Hp-2 (Fig. 1).The distribution of Hp genotypes showed a paucity of the Hp-2-2 (17%) in this adult SCD population with 31% Hp-1-1 and 52% Hp-1-2 genotypes, confirming our previous observations. Hp concentration was strikingly lower in the SCD population (N-48) compared to normal controls (N=3); 2.31 mg/dL vs.45.58 mg/dL, p=0.01. There was no significant difference in Hp concentration among SCD patients with different Hp genotypes (Fig. 2). Similarly Hp levels between patients on and off HU did not differ significantly. Our findings are confirmatory of the data reported recently by Moller et al (Brit. J. Haematol, 153, 105–11, 2011) in 41 children with SCD, who did not find a significant difference in Hp levels in patients on and off HU. To our knowledge this is the first study of Hp levels in adult patients with SCD and different Hp genotypes. A larger study in this patient population looking at Hp levels in relation to both Hp genotype and HU usage is underway. We anticipate that this will provide insights into the role of Hp and its polymorphisms and its contribution to the phenotypic heterogeneity of SCD. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Spontaneous Splenorenal Shunt: An Alternative Cause of Hyperbilirubinemia in Cirrhosis

Purpose: Purpose: To report an alternative cause of hyperbilirubinemia in cirrhosis. Methods: A 47 year old Caucasian male was referred to our GI Clinic for the evaluation of abnormal liver function tests and cirrhosis. His medical history was significant for hypertension treated by lisinopril. He drank 6-8 beers daily for many years, but quit 2 months earlier. He reported persistent jaundice for the previous 6 months, but denied fever or abdominal pain. Physical exam showed mild splenomegaly, otherwise unremarkable. Laboratory data revealed hemoglobin 12 g/dl, MCV 103 fL, platelets 50-70x103/microliter, INR 1.7, B12 and folate normal, AST/ALT were 2xULN, ALP 160 U/L, total bilirubin 6.0 mg/dl with indirect bilirubin 4.1 mg/dl, GGT 20 U/L, haptoglobin <6 mg/dl, and LDH 330 U/L. Ultrasound liver showed splenomegaly (volume 1200cc)and small ascites. Liver biopsy showed cirrhosis with stage 4 fibrosis. Hematology workup failed to find an underlying hemolytic process. CT scan of abdomen showed massive dilation of splenic venous vasculature, suggestive of spontaneous splenorenal shunt, which explained the observed indirect hyperbilirubinemia. Results: The elevation of portal pressure and resistance in cirrhosis leads to an increased gradient between portal pressure and systemic blood flow, potentially initiating portosystemic (PS) shunt formation. The resultant elevation of vascular endothelial growth factor (VEGF) in this setting is crucial for VEGF-dependent angiogenesis for the formation of spontaneous PS shunts. Splenorenal shunt is the most common spontaneous PS shunt in cirrhosis (found in up to 21% of cirrhotics). Esophageal varices are rarely found in the presence of PS shunts. Abdominal CT scan and ultrasound are helpful in diagnosis of spontaneous PS shunt. Hypersplenism in cirrhosis leads to increased hemolysis in the reticuloendothelial system. In the setting of PS shunting, the unconjugated bilirubin will then fail to be cleared by the liver, resulting in the indirect hyperbilirubinemia observed in this case. Spontaneous PS shunts may cause refractory hepatic encephalopathy, intestinal septicemia and Parkinsonism-like features due to manganese deposition in the brain. Thus, identification of spontaneous PS shunt is critical for providing optimal clinical care in cirrhotic patients. Conclusion: Our case indicates that the spontaneous PS shunt (splenorenal being most common) needs to be considered in patients with possible liver cirrhosis who may have indirect hyperbilirubinemia as a predominant clinical feature. An appropriate, expedited workup should then be undertaken.

Leukotriene B4 Mediates Neutrophil Migration Induced by Heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.

Positive HIV ELISA test, autoimmune hemolytic anemia, and generalized lymphadenopathy: A unifying diagnosis

Positive HIV ELISA test, autoimmune hemolytic anemia, and generalized lymphadenopathy: A unifying diagnosis

Fetal and neonatal anemia associated with anti‐Jra: A case report showing a poorly hemolytic mechanism

Although recently published case reports suggest the significance of Jr(a) alloimmunization in the obstetric setting, the involved mechanism still remains unclear. Here we report a case of severe fetal and neonatal anemia associated with anti-Jr(a) alloimmunization, which was successfully managed using Doppler assessment of peak systolic velocity of the fetal middle cerebral artery (MCA-PSV). A Japanese woman with anti-Jr(a) (titer 1024) was referred to our department at 20 weeks' gestation. As fetal MCA-PSV exceeded 1.5 multiple of median, labor was induced and a female neonate of 1998 g was delivered vaginally at 33 weeks and 5 days of gestation. The infant's hematocrit and hemoglobin levels were 25.4% and 82 g/L, respectively, but her total bilirubin level (15 µmol/L; 0.9 mg/dL) and reticulocyte counts (4.5%) were low. During the course, the infant showed no apparent signs of hemolysis. Jr(a) alloimmunization should be recognized as a possible cause of fetal anemia with no direct hemolytic process.

Improved method to evaluate the ability of compounds to destabilize the cellular plasma membrane

In the paper, we present an improved method for evaluation of a compound ability to destabilize erythrocyte plasma membrane. The proposed method is based on the continuous monitoring of the light scattered by erythrocytes exposed to osmotic pressure differences. The kinetics of hemolysis depends on the plasma membrane mechanics and the extent of the osmotic stress. Generally, the osmotic pressure difference of approximately 150 mOsm is taken for measurements, as a result of the equal volume mixing with the physiological salt solutions. In this approach the hemolytic process completion is not established which may result in poor quality and reproducibility of the experimental data. In consequence, inaccurate parameters of the kinetic are determined due to the low quality fitting to the, widely used, single exponential model. In the paper we propose a new experimental protocol allowing to determine the extended set of parameters for kinetics of hemolysis. Namely, the method of the minimal osmotic pressure difference determination is proposed which ensures the completeness of the hemolytic process. This step allows improving the quality and exactness of the calculated parameters. The developed methodology was tested on two qualitatively different, biologically relevant, experiments; evaluation of the peptide effect on the plasma membrane properties and differentiating between human and rabbit erythrocytes.

Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient

Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient

Hemoglobin depletion from plasma: Considerations for proteomic discovery in Sickle Cell disease and other hemolytic processes

Hemoglobin (Hb) depletion with nickel affinity chromatography has been shown to increase the number of proteins identified in proteomic studies of erythrocytes, but limited data exist on the application of this technique in depletion of Hb from plasma or serum required for clinical biomarker studies. The aim of this study was to explore the potential of using nickel-beads for Hb depletion of plasma. Nickel–nitrilotriacetic acid (Ni–NTA) affinity chromatography was used to deplete Hb from hemolyzed plasma samples obtained from children with sickle cell disease (SCD, n=7) and normal human plasma (n=4). Ni–NTA-bound proteins were analyzed by one-dimensional GE, followed by in-gel digestion for characterization using an LTQ-Orbitrap hybrid mass spectrometer. In addition, the loss of two non-Hb-related plasma proteins, thrombospondin1 and L-selectin, by Ni–NTA was determined by ELISA (SCD n=6, non-SCD controls n=2). Ni–NTA resulted in an average 60% decrease in plasma protein concentration, which was not hemolysis dependent. Specifically, Hb (7 peptides) and the top three proteins, -2-macroglobulin (75 peptides), apolipoprotein B-100 (73 peptides), and albumin (42 peptides) were Ni–NTA bound. In addition, using an ELISA assay two non-Hb-associated plasma proteins thrombospondin1 and L-selectin were decreased by Ni-NTA. Hb depletion with Ni–NTA is effective for Hb removal but is not specific. There is a potential for deleterious depletion of potential biomarkers that may limit the applicability of this method. Consideration of alternate methods of Hb depletion for clinical proteomics may be warranted.

Phenotypic Evaluation of a Family Cohort with Hemolytic Anemia

AbstractAbstract 3214 Background:We have identified a kindred in Manitoba and Saskatchewan, Canada, affected by non-immune hemolytic anemia. Red cell morphology, an elevated MCHC and decreased osmotic fragility are consistent with hereditary xerocytosis, a rare hemolytic disorder, for which the causative genetic mutation is unknown. Objectives:To describe the clinical phenotype and inheritance of an uncharacterized chronic hemolytic disorder in a large kindred. Methods:With assistance from each consenting family member, a pedigree was constructed. A focused history was taken and the presence of splenomegaly was assessed by physical examination. Laboratory analysis included a CBC, reticulocyte count, osmotic fragility and peripheral blood film. Biochemical measurements of LDH, ALT, bilirubin, ferritin, haptoglobin, plasma hemoglobin and methemoglobin were performed. Glycolytic enzymes were evaluated in a subset of patients to rule out other rare causes of hemolysis. Results:The family pedigree captured the genetic relations of 342 individuals spanning 5 generations. Consent to participate in the detailed family study was obtained from 137 family members. The average age of the study population was 29 years (range 8 months to 76 years). Laboratory specimens were collected from 26 unrelated spouses and 111 related individuals. Males represented 48% of the studied population. The distribution of reticulocyte counts was distinctly bimodal with no overlap between the two populations, allowing classification of individuals as phenotypically affected or non-affected. The mean percent reticulocyte count of non-affected subjects (related family members and unrelated spouses) was 1.1% (± 0.4, range 0.5–2.3%). Affected subjects had a mean percent reticulocyte count of 9.7% (± 2.6, range 5.3–14.6%). Using this classification, the hemolytic process segregated in an autosomal dominant fashion with complete penetrance. A history of anemia (46 vs. 8%), jaundice (45 vs. 4%), red or brown urine (45 vs. 1%), and either gallstones or cholecystectomy (41 vs. 4%) was more prevalent in affected than unaffected individuals. Episodes of anemia tended to be associated with illness or stress. There was no association between the hemolytic phenotype and neuromuscular, cardiovascular, pulmonary, renal, hepatic, or endocrine disorders. Despite a mean percent reticulocyte count of 9.7% in affected individuals, the mean hemoglobin concentration was not statistically different between affected and unaffected individuals (13.5 ± 1.2 g/dL vs. 13.8 ± 1.4 g/dL, p=0.26). The MCV (96.7 ± 5.5 fL vs. 87.3 ± 5.2 fL, p<0.01) and MCHC (36.6 ± 0.6 g/dL vs. 33.8 ± 0.9 g/dL, p<0.01) were significantly elevated among affected individuals. Morphologically, target cells and stomatocytes were increased among affected individuals. Affected individuals had significantly elevated indirect bilirubin and decreased haptoglobin compared to unaffected or unrelated individuals. Serum ferritin was elevated in all age tertiles in affected individuals compared to non-affected or unrelated individuals, and 7/29 affected individuals had a serum ferritin >900 μg/L. Osmotic fragility performed on 4 affected individuals was decreased. Glycolytic enzymes, screens for unstable hemoglobins and hemoglobinopathies were normal in those tested. Conclusions:In this family study, elevated percent reticulocyte counts were used to characterize the presence of a well compensated, autosomal dominant hemolytic process associated with an elevated MCHC and decreased osmotic fragility. Clinically this condition is associated with gallstones and progressive iron loading. Features are consistent with hereditary xerocytosis. Molecular analysis is currently underway to locate the causative gene and identify the underlying mutation. Disclosures:No relevant conflicts of interest to declare.

Low Level Residual Extravascular Haemolysis Is Commom Following Eculizumab Treatment In Paroxysmal Nocturnal Haemoglobinuria(PNH), but Does Not Affect Transfusion Requirement

AbstractAbstract 4240 Background:PNH is an acquired haemolytic anaemia which is characterised by intravascular haemolysis. The disease is due to a somatic mutation of the PIG-A gene in haematopoietic stem cells resulting in loss (complete or partial) of glycosylphosphatidylinositol-(GPI) anchored proteins on the cell surface. The lack of CD59, one of the GPI-anchored proteins, on the red cell surface leads to increased susceptibility to complement-mediated intravascular haemolysis. Eculizumab is a monoclonal antibody that inhibits the complement protein C5 by preventing its cleavage and leads to transfusion independence in 50% of the patients.Objective: To determine the role of C3 –mediated opsonisation of PNH cells, as we found a significant number of patients treated with Eculizumab still needing transfusion. Results:Twenty six PNH patients treated with Eculizumab and 22 patients from an untreated non transfusion dependant PNH cohort were analysed. Transfusion dependency was present in 14 of 26 patients (54%) compared with transfusion independence in 12/26 (46%) patients. Direct anti-globulin test (DAT) was positive in 21 of 26(81%) patients during eculizumab treatment, while DAT was positive in 2 of 24 untreated patients (8%).All except one patient (20/21) with positive DAT had C3d positivity at varying levels. There was no correlation between the strength of C3d positivity and transfusion dependency.Of the 21 patients who had a positive DAT during eculizumab treatment, samples were available for 16 patients prior to treatment, and 7 of 16 were DAT positive. All the 7 patients were weakly positive for IgG and only one patient had C3d positivity at a low level. The two untreated patients were also only weakly positive for IgG.Among the eculizumab treated patients, 14 of the 21(67%) with a positive DAT test received at least two transfusions compared with no transfusions in the 5 DAT negative patients (p<0.007). There was a reduction in transfusion requirements compared to pre-eculizumab requirement in both DAT positive and DAT negative groups. Interestingly, 7 out of 21(33%) who were DAT positive did not require transfusion post-eculizumab. The median age of the eculizumab treated patient cohort was 51years (range 22–73 years). The median duration of Eculizumab treatment in the 14 DAT positive patients who needed transfusion post eculizumab was 17 months (3.8-98 months) while the median duration of Eculizumab treatment in the 5 DAT negative patients was 7.9 months (1.8-98.5 months). There was no difference in the neutrophil PNH clone size and mean haemoglobin levels (p=0.3), in the DAT positive and negative patients. The median LDH levels did not differ between eculizumab treated DAT positive and negative cases. Conclusion:Eculizumab treatment in PNH patients has resulted in significant reductions in transfusions in a majority of patients, though some patients still require transfusion at a low intensity. The observation of C3 fragments on PNH red cells reveals an extravascular haemolytic pathway which is unmasked by Eculizumab treatment as evidenced by finding new DAT positive tests. The finding of positive DAT test in a 9 of 50(18%) untreated haemolytic PNH patients demonstrates the utility of flow cytometry to analyse PNH clones when clinical suspicion is high, as conventionally finding DAT positivity indicates autoimmune haemolytic process and excludes diagnosis of PNH. The true detection of C3d on red cells causing a positive DAT test post treatment, rather than due to allo-antibodies is important. There is a proportion of patients (33%) who became DAT positive but never needed transfusion indicating the contribution of other factors like bone marrow failure, intercurrent infections in the transfusion dependent DAT positive group.Eculizumab is effective in resolution of intravascular haemolysis, but unmasking of low level C3d mediated haemolysis is an important phenomenon which is not entirely responsible for the persistent transfusion requirement in a subset of patients. Disclosures:No relevant conflicts of interest to declare.

Selective toxin–lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin–membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3 μg/mL and 43.1 μg/mL against sheep and 13.5 μg/mL and 8.8 μg/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore ® technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4 μg/mL to 35.4 μg/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.

Varicella Zoster Virus-induced Hemolytic Crisis in an Infant With Severe Vitamin B 12 Deficiency

Vitamin B 12 deficiency is an uncommon disorder in infancy. Most cases are because of maternal deficiency resulting from insufficient storage and/or reduced intake and are generally seen in exclusively breast-fed infants. Accentuation of the hemolytic process has never been described in association with Varicella Zoster Virus (VZV) infections. We describe a 9-months-old breast-fed infant with megaloblastic anemia secondary to maternal vitamin B 12 deficiency. He presented severe pancytopenia and regression of motor functions and developed hemolytic crisis during a VZV infection. Nutritional cobalamin deficiency should be considered in anemic infants with a history of prolonged exclusive breastfeeding and delayed developmental milestones. VZV infection can trigger a hemolytic process in infants with severe megaloblastic anemia secondary to B12 deficiency. A normal mean corpuscular volume does not rule out megaloblastic anemia, when the condition is combined with severe hemolysis.

Efficacy of Eculizumab in the Treatment of Recurrent Atypical Hemolytic-Uremic Syndrome After Renal Transplantation

Efficacy of Eculizumab in the Treatment of Recurrent Atypical Hemolytic-Uremic Syndrome After Renal Transplantation

Red cell distribution width in chronic heart failure: a new independent marker for prognosis?

Red cell distribution width in chronic heart failure: a new independent marker for prognosis?

In vitro and in vivo haemolytic studies of tentacle-only extract from jellyfish Cyanea capillata

To approach the real haemolytic process of jellyfish toxins, both in vitro and in vivo haemolysis of tentacle-only extract (TOE) from jellyfish Cyanea capillata has been studied. Dose–response curves of the haemolytic activity of TOE in vitro were sigmoid shaped in both erythrocyte suspension and diluted whole blood, with the former more sensitive to TOE. The in vivo haemolysis increased sharply in the first 10min and was followed by a gradual increase in the following 3h, with increasing blood potassium and lactic acid accordingly. SC5b-9 complexes were significantly up-regulated in vitro, but not in vivo. These results showed that the haemolysis of TOE in diluted whole blood and in vivo is not totally consistent with that in the erythrocyte suspension, and blood plasma might play a protective role against haemolysis. Thus we suggested that erythrocyte suspension can be used to test the damage of toxin on erythrocyte membrane, while the diluted whole blood may be more suitable to test the haemolysis of toxins.

Transfusion-related acute lung injury

Sir, Transfusion-related acute lung injury (TRALI) is an uncommon, probably underrecognized complication of transfusion of plasma-containing blood components, which is characterized by acute respiratory distress. It is a clinical diagnosis and may be diagnosed when acute lung injury (ALI) occurs during or within six hours of transfusion in a patient without pre-existing ALI, in the absence of temporally related alternative risk factors for ALI (e.g., sepsis, shock, cardiac failure, etc.).[1] This communication discusses a case of TRALI in a middle-aged woman. A 52-year-old female patient with a diagnosis of inoperable carcinoma of the uterine cervix, received whole blood transfusion, in view of severe anemia. Before blood transfusion, she was alert, oriented, with no cardiorespiratory abnormality on clinicoradiological evaluation. Her vital parameters were normal and total leukocyte count was 8700/mm3 with normal blood biochemistry. After receiving approximately 200 ml of blood for over two hours, she complained of breathlessness, which progressively increased over the next 20 minutes. On examination she was febrile, tachypneic (respiratory rate 32/minute), slightly cyanotic, with a pulse rate of 120/minute, blood pressure 90/60 mmHg, and SpO2 68%. The jugular venous pressure (JVP) was normal and chest auscultation revealed bilateral basal fine inspiratory crackles with normal breath sounds. Sinus tachycardia was noted on an electrocardiogram (ECG). An urgent X-ray chest showed bilateral patchy infiltrates in the mid- and lower zones with normal heart size, suggestive of noncardiogenic pulmonary edema. Echocardiography also ruled out cardiogenic dysfunction. Hematological investigations at this stage showed a total leukocyte count of 3200/mm3, with no evidence of any hemolytic process. In the absence of other mechanisms to explain ALI, a diagnosis of TRALI was made. The blood transfusion was stopped immediately and she was managed with oxygen therapy and intravenous fluids that resulted in a gradual improvement of dyspnea and oxygen saturation. She became completely chest asymptomatic after three days, with a normal chest X-ray. The reported incidence of TRALI in medical literature is one in 5000 transfusions and this may actually be much higher as TRALI is usually an underrecognized and underreported entity.[2] TRALI has been associated with antibodies to WBCs in transfused blood components. Less frequently, it has been associated with leukocyte antibodies in the transfusion recipient. It has also been linked to the infusion of biologically-active lipids in stored cellular blood components.[3] A mild form of TRALI can present with dyspnea and fever, while more severe forms can have severe respiratory distress that can quickly progress to respiratory failure. Other features include hypotension, frothy endotracheal secretions, and so on. Transient leucopenia, as noted in our patient, is occasionally observed and is believed to be due to the sequestration of leukocytes in pulmonary circulation. It is important to distinguish TRALI from volume overload, as the treatment of the two conditions is markedly different. Diuretics are contraindicated in TRALI and there is no role for steroids also. Severe forms of TRALI may require mechanical ventilation.[3] Suspected cases of TRALI should be reported to the blood bank, to permit evaluation and deferral of high-risk donors, especially multiparous females. TRALI can be best prevented by avoiding unnecessary blood-product transfusions in selected populations.

Temperature‐dependent hemolytic activity of membrane pore‐forming peptide toxin, tolaasin

Tolaasin, a pore-forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin-induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 degrees Celsius, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 degrees Celsius and the temperature was decreased, complete hemolysis was observed even at 4 degrees Celsius. These results indicate that tolaasin binding to cell membrane is temperature-sensitive while tolaasin-induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore-forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose-dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin-induced hemolysis can be explained by a multi-hit model.

Unusual Cause Of Severe Anemia In Cirrhosis

Purpose: A 53 year old female with a history of alcohol abuse and cirrhosis presented with progressive leg swelling, fatigue and jaundice. She denied fevers, abdominal pain, melena, confusion, bloody stools or vomiting. Also, she has been sober for the past 8 months. The abnormal physical findings include tachycardia, scleral icterus, hepatomegaly, splenomegaly and 2+ bilateral leg edema. Initial labs showed critically low Hemoglobin 4 gm/dl, elevated indirect bilirubin 6.8 mg/dl and INR of 1.6. Later labs showed low haptoglobin <8 mg/dl [control 43-212], elevated LDH at 308 IU/L [control 98-192], elevated reticulocyte count of 6.2 [control 0.5-1.5], negative hepatitis panel, normal ceruloplasmin and a ferritin of 394. The peripheral smear revealed many burr cells. PRBC transfusion was started at ER. The EGD revealed portal hypertensive gastropathy but no signs of active or old bleeding were noticed. Therefore, the patient's anemia was consistent with the hemolytic process called Spur Cell Anemia seen secondary to End stage liver disease. The patient was treated with blood transfusions. When the hemoglobin was stable around 10 gm% for about 48 hrs she was discharged home. A couple of weeks later patient presented again with severe anemia [Hb 4.6 gm/dl] secondary to hemolysis. Again she was treated with blood transfusions. No GI source of bleeding found. Spur cell anemia is an unusual type of hemolytic anemia in patients with Liver Cirrhosis, particularly in those who abused alcohol. The mechanism of production of these cells has been attributed to the increased Cholesterol: Phospholipids ratio in RBC cell membranes with subsequent remodeling by the spleen because of rigid cell membranes. Therefore, these cells are more susceptible to hemolysis. The spur cell anemia should be suspected in any Cirrhotic patients presenting with hemolysis. The prognosis is extremely poor. They will need repeated blood transfusions and folate replacement. There is no effective treatment. Spur cell anemia may resolve after Liver transplant.Figure: Spur Cells in peripheral smear. [source: eMedicine with permisson from Dr Issam Makhoul]

Cholelithiasis and Cholecystitis

1. Susan Guralnick, MD 1. Stony Brook University Medical Center Stony Brook, NY Biliary Tract Disease in Children. McEvoy CF, Suchy FJ. Pediatr Clin North Am. 1996; 42: 75–98[OpenUrl][1] Cholelithiasis. Friedman JR, Crawford Kennedy M. eMedicine Specialties, Pediatrics: General Medicine, Gastroenterology. 2009. Available at: http://www.emedicine.com/ped/topic381.htm. Accessed June 2009 Cholecystitis. Hebra A, Miller M. eMedicine Specialties, Pediatrics: General Medicine, Gastroenterology. 2008. Available at: http://emedicine.medscape.com/article/927340-overview. Accessed June 2009 Gallbladder Disease. O'Neill J, Grosfeld J, Fonkalsrud E. In: Principles of Pediatric Surgery. 2nd ed. St. Louis, Mo: Mosby; 2004 Cholelithiasis is an uncommon occurrence in healthy children, with an incidence ranging from 0.15% to 0.22%. The condition is seen more frequently in children who have certain predisposing conditions. Cholelithiasis can occur at any age, including prenatally, but is most common during puberty. Females are at significantly higher risk, with an overall 4:1 female-to-male predominance that increases to 11 to 22:1 during adolescence. Gallstones form when bile is supersaturated with cholesterol or bilirubin to the point that the substances no longer are soluble. This effect, along with bile stasis, allows the cholesterol or calcium bilirubinate crystals to remain in the gallbladder (or ductal system for brown stones) long enough to … [1]: {openurl}?query=rft.jtitle%253DPediatr%2BClin%2BNorth%2BAm.%26rft.volume%253D42%26rft.spage%253D75%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx