Fragmented Red Blood Cells Research Articles

Bacteria can promote calcium oxalate crystal growth and aggregation

Our previous report showed that uropathogenic bacteria, e.g., Escherichia coli, are commonly found inside the nidus of calcium oxalate (CaOx) kidney stones and may play pivotal roles in stone genesis. The present study aimed to prove this new hypothesis by direct examining CaOx lithogenic activities of both Gram-negative and Gram-positive bacteria. CaOx was crystallized in the absence (blank control) or presence of 10(5) CFU/ml E.coli, Klebsiella pneumoniae, Staphylococcus aureus, or Streptococcus pneumoniae. Fragmented red blood cell membranes and intact red blood cells were used as positive and negative controls, respectively. The crystal area and the number of aggregates were measured to initially screen for effects of bacteria on CaOx crystal growth and aggregation. The data revealed that all the bacteria tested dramatically increased the crystal area and number of crystal aggregates. Validation assays (spectrophotometric oxalate-depletion assay and an aggregation-sedimentation study) confirmed their promoting effects on both growth (20.17±3.42, 17.55±2.27, 16.37±1.38, and 21.87±0.85% increase, respectively) and aggregation (57.45±2.08, 51.06±5.51, 55.32±2.08, and 46.81±3.61% increase, respectively) of CaOx crystals. Also, these bacteria significantly enlarged CaOx aggregates, with the diameter greater than the luminal size of distal tubules, implying that tubular occlusion might occur. Moreover, these bacterial effects were dose-dependent and specific to intact viable bacteria, not intact dead or fragmented bacteria. In summary, intact viable E.coli, K.pneumoniae, S.aureus, and S.pneumoniae had significant promoting effects on CaOx crystal growth and aggregation. This functional evidence supported the hypothesis that various types of bacteria can induce or aggravate metabolic stone disease, particularly the CaOx type.

Pitfalls in the diagnosis of hepatic epithelioid hemangioendothelioma by FNA and needle core biopsy

Epithelioid hemangioendothelioma (EHE) of the liver is an extremely rare tumor that masquerades as an epithelial neoplasm and poses significant diagnostic pitfalls for the cytopathologist. This report describes a 64-year-old woman whom on computerized tomography was found to have multiple peripherally calcified hypodense lesions throughout the liver and an ipsilateral adrenal mass. Fine needle aspiration and needle cores biopsies of the largest liver lesion showed epithelioid cells and spindle cells in fibrous stroma. The epithelioid cells had round or oval, hyperchromatic nuclei with smooth nuclear contours, delicate cytoplasm and indistinct cytoplasmic borders. Rare cells had intracellular vascular lumen containing fragmented or intact red blood cells. The cells were individually dispersed or arranged in nests and vague tubular arrays. No mitotic figures or necrosis were seen. An immunohistochemical profile demonstrated diffuse CD31 and focal CD 34 and nuclear Fli-1 immunoexpression and low Ki-67 proliferative activity (1%) within lesional cells confirming the diagnosis of EHE. The differential diagnosis of EHE which includes cholangiocarcinoma, hepatocellular carcinoma, metastatic carcinoma and melanoma is discussed.

Identification of Spurious Hemolysis in Anticoagulated Blood with Sysmex XE-2100 and Siemens Advia 2120

Various degrees of hemolysis might occur during collection, processing and storage of blood bags or blood tubes for hematological testing. In both circumstances, the identification of hemolysis is challenging since the centrifugation process is not required. The aim of this study was to identify simple hematological parameters that would help identify the presence of hemolysis in anticoagulated blood. Twenty tubes containing K2EDTA anticoagulated blood were randomly selected from outpatient samples and divided in two aliquots. The former was immediately analyzed, whereas the latter was subjected to mechanical hemolysis by aspirating whole blood two times through a very fine needle to generate mechanical hemolysis. Both aliquots were tested on Advia 2120 and Sysmex XE-2100. The double aspiration of the blood through the fine needle caused a remarkable hemolysis with significant decrease of red blood cell (RBC) count (-17 +/- 11%; p < 0.01), hematocrit (-18 +/- 12%; p < 0.01) and reticulocytes (-24 +/- 13%; p < 0.01), but not of hemoglobin, white blood cell or platelet counts. A remarkable increase of immature platelet fraction (IPF) on XE-2100 and RBC ghosts on Advia 2120 was observed in the hemolyzed samples, whereas RBC fragments did not vary significantly. A significant correlation was also observed between hemolysis and reticulocyte count (r = 0.823; p < 0.01), IPF (r = -0.502, p = 0.024) and RBC ghosts (r = -0.711; p < 0.01). Receiver Operating Characteristic (ROC) curve analysis demonstrated a good performance of both IPF and RBC ghosts (i.e., AUCs 0.91 and 0.96, respectively) for distinguishing non-hemolyzed from hemolyzed specimens. The absolute values of both IPF and RBC ghosts perform efficiently for distinguishing hemolyzed from non-hemolyzed specimens, although neither reached 100% sensitivity and specificity. Nevertheless, the demonstration that both parameters significantly increase after hemolysis can be reliably used to distinguish hemolyzed from non-hemolyzed blood.

Advancements in laboratory diagnostics: an invaluable tool for assessing quality of blood transfusions.

The interesting article by Willy A. Flegel that recently appeared in this journal provides valuable insights into an ongoing debate on the use of fresher material for blood transfusions1. Regardless of important considerations about the delicate balance between selection of units and blood supply, an innovative approach for defining the quality of transfused components is increasingly supported by recent advancements in laboratory diagnostics, which would enable to determine whether leucocytes, platelets and red blood cells are suitable for transfusion regardless of the ageing of the stored material. Several examples can be given, whereby routine, automatic flow haemocytometry combined with additional technical tools provides valuable information. As regards platelets, the loss of function during storage is mirrored by disc-to-sphere transformation, extension of pseudopodia, alteration of the canalicular system and granules, up to loss of storage granules2. Some haemocytometers already provide a variety of activation-related information about these blood elements. For example, in Advia 2120 (Siemens Diagnostics, Tarrytown, New York, United States of America), the intensity of light scattered by platelets is assessed at two different angles (i.e., 2 to 3° and 5 to 15°). Mean platelet volume and mean platelet component are then calculated and from these paired measures. The latter parameter, in particular, has been shown to be a reliable index of platelet degranulation, since it is directly related to CD62 expression, which is highly indicative of platelet activation and ageing3. The same instrument, along with others such as the PENTRA 60C (Horiba-ABX Diagnostics, Montpellier, France), provide specific flags or alerts in the presence of degranulation of polymorphonuclear leucocytes4, which is a hallmark of functional impairment5. More specifically, the Advia 2120 also enables reliable quantification of the number of degranulated neutrophils in specific clusters of the peroxidase scattergram based on cell volume and intracellular myeloperoxidase concentration. Additional morphological changes that characterise gradual deterioration of leucocytes in stored blood include a more homogeneous staining of nuclei, the separation of the nuclear lobes or irregular lobulation and vacuolisation, which can be reliably identified in the scattergram of modern haemocytometers such as those of the Sysmex XE series, in which the intensity of side scatter is dependent on the complexity of nuclear lobulation and granules within the cytoplasm6. It is also noteworthy that several changes occur to red blood cells during medium- and long-term storage in blood bags, including reduced deformability, altered adhesiveness and aggregability, and decreased 2,3-diphosphoglycerate and ATP content, to the point of frank injury and spurious haemolysis7. Specific macromolecular remodelling, along with lipid peroxidation and/or oxidative damage to the membrane, are mainly responsible for the onset of irreversible morphological abnormalities and loss of function. Most of these physicochemical changes, usually referred to as the “storage lesion”, can be identified by time-consuming or expensive analytical techniques such as flow cytometry, time-resolved phosphorescence anisotropy and microscopy, which are unsuitable for rapid examination of a large number of blood bags8. Nevertheless, the presence of red blood cell crenation or fragmentation -and finally haemolysis- can now be reliably and rapidly identified by specific erythrocyte indices on automated flow haemocytometers, such as the “immature platelet fraction” on XE-2100 (Sysmex, Kobe, Japan), or “RBC ghosts” on Advia 21209. Another interesting approach is ThromboLUX™ (LightIntegra Technology Inc., Vancouver, British Columbia, Canada), which uses the principle of dynamic light scattering -also known as photon correlation spectroscopy or quasi-elastic light scattering- to identify and quantify the particles that may be present in a platelet concentrate, as well as showing how they respond to temperature stress. The instrument finally generates a quality score on a scale ranging from 0 to 40, with platelet concentrates scoring from 13 to 40 containing mostly discoid and functional platelets. Recent data show that the information from ThromboLUX™ correlates adequately with that from flow cytometry and microscopy and may, therefore, be suitable for quality assessment of platelet concentrate samples10. Several technical advances have occurred in laboratory diagnostics over the past decades. These have not only improved the global quality of testing, but have also enabled reliable application to transfusion medicine for the assessment of the quality of transfused components, as attested by some of the examples previously provided. The modern, fully-automated haemocytometers -which are characterised by high accuracy and precision, notable throughput and full integration with information systems- can also provide a kaleidoscope of cellular indices. Most of these novel parameters are still partially unexploited, but it is conceivable that they may be reliably used to detect artefactual changes in leucocytes, platelets and red blood cells, which may be suggestive of less functional, defective or even apoptotic cells, and finally establish as to whether cellular elements within blood bags are still viable for transfusion. Future studies should be focused on the evaluation of the usefulness of these parameters in the setting of transfusion medicine.

Fatal Thrombotic Microangiopathy Developing within 24 Hours of Carfilzomib in a Patient with Relapsed Multiple Myeloma (MM)

Abstract 5037Thrombotic Microangiopathy (TMA) has been reported in several clinical settings including viral infection, following drug exposure, and in patients with cancer including three with MM treated with bortezomib. TMA can present with the pentad of microangiopathic hemolytic anemia (MAHA), thrombocytopenia, acute renal insufficiency, neurologic abnormalities and fever. Cardiac involvement, when present, is associated with poor prognosis.We herein present the first case of TMA occurring in association with Carfilzomib. The patient was a 62 year old African American female with progressive IgG lambda MM by serum markers (lambda light chains 41. 8 mg/dl and IgG 2230 mg/dL), imaging studies with extramedullary disease involving the muscular tissue and 20% bone marrow plasmacytosis with complex cytogenetics. Her last therapy consisted of melphalan-based autologous transplant 8 months earlier. Chronic thrombocytopenia was present likely due to therapy related myelodysplastic syndrome (T-MDS). Peripheral smear at relapse showed no evidence of circulating blasts, plasmacytosis or schistocytosis.The patient received a 20 mg/m2 IV push single dose of carfilzomib (CFZ), a novel proteasome inhibitor, on 12/03/2011. Within 24 hours of the CFZ dose, she developed clinical and laboratory evidence of TMA including increasing lethargy, fever to 101 F, renal dysfunction (creatinine 0. 8 to 2. 7 mg/dL), worsening thrombocytopenia (29, 000 to 16,000/μL), and MAHA with hemoglobin dropping (9. 3 to 7. 9 g/dL), progressive bilirubinemia, undetectable serum haptoglobin level and a 5-fold increase in serum LDH (from 256 to 1200 IU/L). Peripheral smear confirmed the presence of schistocytes. No other causes for TMA could be identified; ADAMTS 13 levels were 52%, with negative results for heparin antibodies, anti-phospholipid antibodies, direct coombs and PCR for Adenovirus, Parvovirus, CMV and Human herpes virus 6. Coagulation studies were normal. Renal ultrasound showed no obstruction. The left ventricular ejection fraction by echocardiogram on 12/19/11 was 25% compared to 60% on prior MUGA scan and echocardiogram two and nine months before CFZ.She failed to respond to various agents including corticosteroids and therapeutic plasmapheresis and died 44 days after the single CFZ dose. Repeat serum myeloma markers showed response to CFZ. Autopsy examination revealed TMA with presence of red blood cell fragments and multiple fibrin thrombi in the kidneys without evidence of amyloidosis or plasma cell infiltration. Aggregates of CD 138 positive plasma cells were present in the visceral pleura of the stomach, bladder, pancreas and intestines but without organ infiltration. The bone marrow was hypercellular with 5% involvement with lambda predominant plasma cells. At death, the serum levels of lambda light chains and IgG were 54. 8 and 1000 mg/dL, respectively.This case describes the abrupt onset of ultimately fatal TMA within 24 hours of a single dose of 20 mg/m2 of CFZ. No other cause for TMA could be identified despite an extensive work-up. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Renal injury due to anti-glomerular basement membrane antibody-mediated glomerulonephritis without circulating antibody.

Renal injury due to anti-glomerular basement membrane antibody-mediated glomerulonephritis without circulating antibody.

Evaluation of schistocytes measurement guidelines

The schistocytes are fragmented red blood cells mainly observed in the setting of hemolytic anemias and particularly among the thrombotic microangiopathies. The presence of schistocytes is an important criterion for the diagnosis of mechanical anemias, though the identification of these cells remains problematic. As a high variability of the morphologic identification criteria of the schistocytes among morphologists has been observed, some guidelines have been proposed after workshops (French and Italian groups). The International council for standardization in hematology published a consensus in November, 2011. The French group of cellular hematology (GFHC) aimed to recover the opinion of French biologists directly confronted to schistocytes measurements. 169 out 500 (34%) answered 10 questions dealing with the identification and measurements of schistocytes as proposed by the ICSH guidelines. A consensus was reached for the urgent need of guidelines documents, moreover in the current background of the European accreditation NF EN ISO 15189 rules. A traduction in native (French) language as warmly wished in order to facilitate the diffusion of the information. New fragmented red cell parameter recently provided by 2 manufacturers of automated blood cell counters remained doubtfull for routine use for half of the biologists.

Thrombotic Microangiopathies

Thrombotic microangiopathy results from thrombotic occlusion of the microvasculature leading to fragmentation of red blood cells, profound thrombocytopenia, and a microangiopathic hemolytic anemia with elevation of lactate dehydrogenase and negative direct Coomb's test. This constellation of clinical and laboratory findings is not due to one disease entity; rather, it represents a variety of underlying diagnoses. Among the major disease entities are TTP/HUS, which can be congenital or acquired, bacterial infections, medications, vascular or endothelial pathology like Kasabach-Merritt phenomenon, and stem cell transplantation. In this paper, we offer a review of some of the major causes of thrombotic microangiopathy.

A case of hereditary pyropoïkilocytosis with mild expression and delayed onset

We report on a case of hereditary pyropoïkilocytosis fortuitously diagnosed in a 34-year old woman issued from Benin. Laboratory tests indicated a moderate haemolytic anaemia with a marked microcytosis. Blood film examination revealed a striking anisopoikilocytosis characterized by elliptocytes, numerous red blood cells (RBC) fragments and microspherocytes. The histogram of RBC volume distribution showed two populations of RBC: a normocytic and a very microcytic population, this later corresponding to the RBC fragmentation. These features strongly suggested a membrane disorder, particularly an hereditary pyropoïkilocytosis (HPP). The thermal unstability of the cytoskeleton was demonstrated by enhanced red cell fragmentation after in vitro exposure to heat which occurs at a lower temperature as compared to normal red cells. The diagnosis of HPP was confirmed by specialized investigations (osmotic gradient ektacytometry and erythrocytic membrane proteins electrophoresis). HPP is considered as a severe form of hereditary elliptocytosis characterized by jaundice and a severe haemolytic anaemia which usually appears during the neonatal period and the childhood. Our report is intriguing because of the delayed diagnosis of HPP in a patient who presented moderate clinical manifestations.

Thrombotic Microangiopathies, Thrombotic Thrombocytopenic Purpura, and ADAMTS-13

Thrombotic microangiopathy (TMA) is a term used to describe a group of disorders characterized by hemolytic anemia (with prominent red blood cell fragmentation), thrombocytopenia, and thrombosis in the microvasculature. It may be used when describing patients with thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome, atypical hemolytic uremic syndrome, as well as a myriad of other disorders in which the TMA may be secondary to another disease or disorder. While limited information exists as to the exact cause of microthrombosis in many TMA, recent advances have been made in the understanding of TTP and its pathophysiology. This progress can be attributed to discovery of the von Willebrand factor cleaving protease ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), whose absence in TTP has given the disorder a distinct molecular identity. The discovery of this metalloprotease has prompted a significant amount of research relating to its role in TTP as well as its general function in hemostasis. The exact mechanisms by which this metalloprotease achieves its role are slowly being understood and these now provide other avenues by which TMA may occur.

Letter to the editor Cyclophosphamide-rescued plasmapheresis-unresponsive secondary thrombotic thrombocytopenic purpura caused by Sjögren’s syndrome

Letter to the editor Cyclophosphamide-rescued plasmapheresis-unresponsive secondary thrombotic thrombocytopenic purpura caused by Sjögren’s syndrome

A simple and efficient method for the preparation of live leukocytes from peripheral blood using the LeukoCatch&amp;lt;sup&amp;gt;TM&amp;lt;/sup&amp;gt; system

Leukocytes from peripheral blood (PB) are of great value for diagnosis as well as basic and clinical research. However, no easy, centrifugation-free method is available for the isolation of live leukocytes from blood. We here develop a simple and quick method for the purification of viable leukocytes from whole blood using novel tools, named tLeukoCatch (tip-type) or sLeukoCatch (syringe-type), which is equipped with three Pall filter layers and captures leukocytes but not red blood cells (RBCs) in whole blood. Indeed, we showed that several million leukocytes per mL (~35% of the recovery rate) were captured and eluted from whole blood. The number of contaminant RBCs decreased from several million to several thousand. When mouse blood was hemolysed, almost all of the lysed RBC fragments were removed by passage through sLeukoCatch. Optical microscopic observation confirmed that the recovered leukocytes were sufficiently healthy to respond to growth stimuli. Efficient leukocyte recovery was also confirmed for hemolysed human blood. These results suggest that the LeukoCatchTM system is useful for bedside diagnosis and basic research with blood samples.

ICSH recommendations for identification, diagnostic value, and quantitation of schistocytes

Schistocytes are fragments of red blood cells (RBCs) produced by extrinsic mechanical damage within the circulation. The detection of schistocytes is an important morphological clue to the diagnosis of thrombotic microangiopathic anemia (TMA). Reporting criteria between different laboratories, however, are not uniform, owing to variability of shape and nature of fragments, as well as subjectivity and heterogeneity in their morphological assessment. Lack of standardization may lead to inconsistency or misdiagnosis, thereby affecting treatment and clinical outcome. The Schistocyte Working Group of the International Council for Standardization in Haematology (ICSH) has prepared specific recommendations to standardize schistocyte identification, enumeration, and reporting. They deal with the type of smear, method of counting, morphological description based on positive criteria (helmet cells, small, irregular triangular, or crescent-shaped cells, pointed projections, and lack of central pallor). A schistocyte count has a definite clinical value for the diagnosis of TMA in the absence of additional severe red cell shape abnormalities, with a confident threshold value of 1%. Automated counting of RBC fragments is also recommended by the ICSH Working Group as a useful complement to the microscope, according to the high predictive value of negative results, but worthy of further research and with limits in quantitation.

Hämolytische Anämien beim Erwachsenen

The erythrocyte lifespan in haemolytic anemia is shortened while erythropoesis is increased. Important labaratory findings are increased reticulocytes, LDH, indirect bilirubin and a decreased haptoglobin level. The most important diagnostic tool for further work up of hemolytic anemia is the direct antiglobulin test (DAT, Coombs test) to differentiate autoimmune hemolytic anemia (AIHA) from other causes. Another important group are fragmentation syndroms (hemolytic uremic syndrome and thrombotic thrombocytopenic purpura). In these forms of haemolytic anemia fragmented red blood cells can be found in the blood smear together with thrombocytopenia. A severe problem in paroxysmal nocturnal hematuria is the incidence of thrombosis. The following review describes the most important forms of hemolytic anemia in the adult and the diagnostic and therapeutic strategies.

Thrombotic thrombocytopenic purpura complicating immune thrombocytopenic purpura—A case report

A 60-year-old male was evaluated for episodes of gingival bleed. Past medical history was significant for hypertension currently treated with clonidine and aliskiren and early stage prostate cancer. Social and family histories were noncontributory. Review of systems was negative except for bleeding. Physical examination was unremarkable. Platelet count was 8 × 109/l and peripheral smear demonstrated thrombocytopenia with few large platelets. Immune Thrombocytopenic Purpura (ITP) was diagnosed, and the patient was treated with prednisone 40 mg/day with significant improvement in his platelet count. Prednisone was then tapered to 5 mg/day. Six months later, the patient developed gingival bleed, chest pain, shortness of breath, and hematuria. Physical examination revealed elevated blood pressure (200/104 mm of Hg), gingival bleeding, and a hematoma of the tongue. Laboratory tests showed platelet count 6 × 109/l, Lactate Dehydrogenase (LDH) 1348 u/l, Antinuclear Antibody (ANA) titers 1:160, and total bilirubin 6 mg/dl. Coagulation profile, hepatitis panel, Thyroid-stimulating hormone (TSH), Human immunodeficiency virus (HIV), Rh factor, and direct antiglobulin test were negative. The diagnosis of ITP, myocardial infarction, and uncontrolled hypertension was made. Prednisone was increased to 60 mg/day, and blood pressure control was optimized. On admission Day 2, the patient developed altered mental status and fever. Computerized Axial Tomography (CAT) scan of head revealed intracranial bleed. His hemoglobin level fell by 2 g/dl and serum creatinine increased. Peripheral smear confirmed the diagnosis of Thrombotic Thrombocytopenic Purpura (TTP). Plasmapheresis was initiated, mental status returned to baseline, and renal function remained stable. ADAMTS -13 (Is a metalloproteinase also known as von Willebrand factor-cleaving Protease) activity obtained before plasmapheresis was 12%, and von Willebrand factor cleaving inhibitor level was 1.4 (normal less than 0.4). Plasmapheresis was continued for 12 exchanges with modest recovery of platelet count from. The patient remained anemic, LDH levels remained high, and 2+ schistocytes persisted on peripheral blood smear. Therapy with rituximab (4 weekly doses of 375 mg/m2), methylprednisone (1 mg/kg/day) and intravenous immunoglobulin G was initiated. After the fourth course of this regimen, the platelet count was 107 × 109/L, LDH levels decreased, the hematocrit remained stable, and peripheral smear showed no fragmented red blood cells. The patient was successfully tapered off prednisone and has remained well. TTP and ITP existing in the same patient are uncommon. Seventeen cases have been reported in the literature with both conditions occurring at different times or concurrently (only one case). Baron et al. [1] reviewed 11 cases of TTP, six of which had an underlying autoimmune disorder. Baron suggested that these six cases represented a distinct clinical syndrome, “mixed immune thrombocytopenia,” due to disturbed immune regulation. Our patient initially presented with isolated thrombocytopenia and positive ANA titers suggestive an immune-mediated disease that responded transiently to steroids. On readmission, he was diagnosed with TTP and started on appropriate therapy with good clinical outcome. In conclusion, emphasis should be placed on a thorough re-evaluation of each recurrence of thrombocytopenia. Overlapping of these two distinct clinical entities may be misleading and can delay appropriate treatment. As evidenced by existing literature, the patient who presents with an initial episode of either ITP or TTP, may subsequently present with either of these syndromes at relapse. Avani Changela*, Kuppuswamy Jagarlamudi , Gina Villani , Meena Alhuwalia , Ashraf Elnawawi , * The Brooklyn Hospital Center, Internal Medicine, Brooklyn, New York 11201, The Brooklyn Hospital Center, Hematology and Oncology, Brooklyn, New York 11201, The Brooklyn Hospital Center, pathology, Brooklyn, New York 11201.

Kidney diseases beyond nephrology: intensive care

Sodium bicarbonate has been used for several years for its presumed nephroprotective effects in several settings. In particular, its role in cardiac surgery-associated acute kidney injury (CSA-AKI) has been explored. CSA-AKI is a particular type of cardio-renal syndrome for which no clear understanding of pathogenesis exists and no proven effective prophylaxis or treatment has as yet been established [1]. Previous studies have reported injury to red cells and release of free haemoglobin during cardiopulmonary bypass (CPB) [2]. Besides complete red blood cell fragmentation (induced by CPB, valvular prosthesis, transfusion reactions or genetic defects predisposing to reduced erythrocyte membrane stability), there can also be sublethal red cell damage, resulting in altered rheological properties. Increased levels of free red blood cell constituents, together with an exhaustion of their scavengers, transferrin and haptoglobin, result in a variety of serious clinical consequences, such as increased systemic vascular resistance, altered coagulation prof ile, platelet dysfunction, renal tubular damage and increased mortality. Such injury raises concerns that CSA-AKI may be a form of renal sideropathy and that free or inappropriately liganded iron-related toxicity may play a role [3]. Free iron has the potential to catalyse the formation of super-oxide radical and hydrogen peroxide yielding hydroxyl radical. It is known that an acid environment typical of tubular urine enhances the formation of reactive hydroxyl radicals. Finally, tubular obstruction may allow greater time for endocytotic uptake of free haemoglobin into proximal tubular cells, which is associated with proximal tubular cell necrosis. Oxidative stress has been shown to play a key role in the development of toxic and ischaemic AKI. Interestingly, urinary alkalinization with sodium bicarbonate might protect from the pathophysiological mechanisms causing CSA-AKI. A recent double-blind randomized controlled trial showed that bicarbonate might attenuate CSA-AKI, potentially directly affecting iron-related toxicity, as indicated by a smaller increase in urinary biomarker neutrophil gelatinaseassociated lipocalin (NGAL) [4]. At neutral or alkaline pH, free ferric ions precipitate as insoluble ferric hydroxide, which is excreted as inert complex in the urine. More alkaline urine reduces the generation of injurious hydroxyl radicals and lipid peroxidation. Bicarbonate directly scavenges hydroxyl ions and, as a not well-adsorbable anion compared with chloride, causes more rapid volume excretion and thereby reduces the contact time between injurious radicals and renal tubules. Peak plasma creatinine increases were typical by the third post-operative day regardless of the study group. Furthermore, exposure to sodium bicarbonateattenuated renal oxidative stress as reflected by lower urinary NGAL levels and urinary NGAL/creatinine ratios. As expected, exposure to sodium bicarbonate infusion was associated with a higher plasma pH, higher plasma bicarbonate and urinary alkalinization. Fewer patients in the sodium bicarbonate group (16 of 50) developed a post-operative increase in serum creatinine compared with control (26 of 50) (P = 0.043). The increase in plasma creatinine, plasma urea, urinary NGAL, and urinary NGAL/urinary creatinine ratio was less in patients receiving sodium bicarbonate. As the authors acknowledge, bicarbonate apparently preventedonly mild renal dysfunction which may have limited effects on clinical outcome. Furthermore, the potential clinical risks of systemic alkalinization, e.g. oxyhaemoglobin dissociation curve shifts resulting in higher oxygen–haemoglobin affinity with possible relative tissue hypoxaemia, have not been fully evaluated in this pilot trial. The effects of bicarbonate on higher risk cardiac surgery patients remain to be evaluated. These results suggest finally a novel utility of urinary NGAL measurements not as diagnostic biomarker but as monitor of the efficacy of therapy for AKI. Sodium bicarbonate has also been used for nephroprotection in contrast-induced acute kidney injury (CI-AKI). The benefit of CI-AKI prophylaxis has mainly been shown for patients with reduced glomerular filtration rate (GFR) (<50 mL/min/1.73 m), heart failure or diabetes, and/or patients with high risk scores (Thakar score [5] or Mehran score [6]). In this light, intensive care unit (ICU) patients undergoing angiography should always be considered as high-risk patients. The International Consensus Conference in Intensive Care Medicine recently considered the

Rapid Identification of Thrombocytopenia-Associated Multiple Organ Failure Using Red Blood Cell Parameters and a Volume/Hemoglobin Concentration Cytogram

Thrombocytopenia-associated multiple organ failure (TAMOF) has a high mortality rate when not treated, and early detection of TAMOF is very important diagnostically and therapeutically. We describe herein our experience of early detection of TAMOF, using an automated hematology analyzer. From 498,390 inpatients, we selected 12 patients suspected of having peripheral schistocytosis, based on the results of red blood cell (RBC) parameters and a volume/hemoglobin concentration (V/HC) cytogram. We promptly evaluated whether the individual patients had clinical manifestations and laboratory findings were consistent with TAMOF. Plasma exchanges were then performed for each patient. All 12 patients had TAMOF. The mean values of RBC parameters were significantly higher in all of the patients than with the reference range, however, 3 patients had % RBC fragments within the reference range. The mean value of ADAMTS-13 activity was slightly lower in patients compared with the reference range. Of the 12 patients, remission was obtained in 9 patients (75%) within 4 to 5 weeks using plasma exchanges. Three patients died. An increased percentage of microcytic hyperchromic cells with anisocytosis and anisochromia indicated the presence of schistocytes, making it an excellent screening marker for TAMOF. Identification of TAMOF with RBC parameters and a V/HC cytogram is a facile and rapid method along with an automated hematology analyzer already in use for routine complete blood cell counting test.

Pseudo-thrombotic thrombocytopenic purpura: A rare presentation of pernicious anemia

Schistocytes are fragmented red blood cells due to the flow of blood through damaged capillaries and indicate endothelial injury. They are typical of microangiopathic hemolytic anemia seen in life threatening conditions like disseminated intravascular coagulation or thrombotic thrombocytopenic purpura/hemolytic uremic syndrome .We report a rare sub-acute presentation of pernicious anemia with hemolysis, thrombocytopenia and numerous schistocytes that was initially diagnosed as a more serious thrombotic thrombocytopenic purpura.A 31-year-old Caucasian woman presented with fatigue and paresthesia of both feet for 1 week. Past medical history included hypertension and gastro-esophageal reflux disease. Examination revealed scleral icterus and pallor. Examination of the abdomen did not show hepatosplenomegaly. Initial laboratory tests showed severe anemia, and low platelets. Indirect bilirubin and serum Lactate De Hydrogenase were elevated. Prothrombin time, partial thromboplastin time, serum fibrinogen, and serum fibrin degradation product levels were normal. Peripheral smear revealed numerous schistocytes, anisocytosis and macro-ovalocytes. Thrombotic thrombocytopenic purpura (TTP) was suspected due to the constellation of sub-acute onset of fatigue and paresthesia along with thrombocytopenia, schistocytes and an elevated LDH. Plasmapheresis was initiated for possible TTP. However, platelet count worsened despite plasmapheresis for 4 days. On re-evaluation, vitamin B(12) was found to be low. Treatment with intra-muscular vitamin B(12) led to symptomatic and hematologic improvement. Pernicious anemia was confirmed by the presence of anti-intrinsic factor antibodies, elevated serum gastrin level and atrophic gastritis.Clinicians must be aware of unusual clinical presentation of vitamin B(12) deficiency with schistocytes as the management is simple and effective.

Circulating Nucleosomes Reflect Disease Activity in Patients with Thrombotic Microangiopathies.

AbstractAbstract 1437 Background:Thrombotic microangiopathies (TMAs) including thrombotic thrombocytopenic purpura (TTP) associated with acquired or congenital severe ADAMTS13 deficiency, typical diarrhea-associated (D+) and atypical (D-) hemolytic uremic syndrome (HUS), and other TMAs associated with disseminated neoplasia, some drugs, pregnancy and several infectious or autoimmune conditions are life-threatening diseases characterized by microvascular thrombosis with organ dysfunction, consumptive thrombocytopenia and hemolytic anemia with red blood cell fragmentation. Severe autoantibody-induced ADAMTS13 deficiency is found in about 60% of patients diagnosed with acquired TTP and severe constitutional ADAMTS13 deficiency due to ADAMTS13 mutations is found in almost all patients with hereditary TTP. However, some patients with severe acquired or hereditary ADAMTS13 deficiency may be asymptomatic for prolonged time intervals and some triggering factor(s) may be needed to bring about acute disease. Extracellular exposure of DNA and histones is a recently discovered mechanism linking inflammation with thrombosis. Chromatin fibers released from activated neutrophils (Neutrophil Extracellular Traps, NETS) bind platelets, erythrocytes and DNA/histone complexes stimulate platelet aggregation and coagulation. We investigated stored plasma samples from patients with various forms of TMA, both during acute disease and during remission, for circulating nucleosomes, i.e. subunits of chromatin composed of DNA wrapped around a histone core. Patients:First, 29 patients with acute TMA (6 acquired TTP with severe ADAMTS13 deficiency, 4 D+ HUS, 8 neoplasia-associated TMAs and 11 TMAs of undetermined etiology) were investigated and compared to 10 healthy controls. Second, serial samples from 8 patients with TTP and acquired ADAMTS13 deficiency obtained during acute disease, during plasma exchange (PE) treatment and during remission were analyzed as were samples from 3 hereditary TTP patients during acute disease and during remission. Methods:Plasma DNA was quantified, nucleosomes were measured using an antibody against DNA complexed with histones H2A/H2B. Plasma lactate dehydrogenase and myeloperoxidase were assayed using commercial kits. Results:Significantly elevated levels of DNA and histones organized in nucleosomes were found in patients with acute TMAs. Increase in nucleosome levels was paralleled by elevation of plasma myeloperoxidase and calgranulin pointing to neutrophils as the origin of nucleosomes. All patients achieving clinical remission from acute acquired TTP showed normalized levels of nucleosomes indistinguishable from those in healthy controls. Transient disease flare-ups with worsening platelet counts in acute acquired TTP patients subjected to daily PE therapy were often associated with a transient increase of the nucleosome levels. One patient who died after 10 days of PE had extremely high nucleosome levels prior to death. Three patients with hereditary severe ADAMTS13 deficiency had variably elevated nucleosome levels during active disease and normal levels in remission maintained by regular plasma infusion (n=2) or mildly increased levels in remission without maintenance plasma therapy (n=1). Conclusion:Circulating nucleosomes may reflect the degree of systemic inflammation and seem – at least in some cases - to reflect the second hit precipitating acute disease in patients at risk for acute TMA, such as those with acquired or hereditary severe ADAMTS13 deficiency. Disclosures:Kremer Hovinga:Baxter Bioscience: Consultancy. Lämmle:Baxter Bioscience: Consultancy.

Severe thrombosis and acute thrombocytopenia in a cancer patient

A 62-year-old Caucasian female who had recently been diagnosed with multiple myeloma with a sacral plasmacytoma received induction treatment with bortezomib, cyclophosphamide, and dexamethasone. She had a significant response to therapy and had just begun the process of mobilization and collection of peripheral blood progenitor cells for autologous stem cell transplantation when she presented to the emergency center (EC) with pain and swelling of the left upper extremity. Her blood pressure on presentation was 114/69 mmHg with a heart rate of 98 beats per minute. Serum sodium was 136 mEq/L, potassium 4.3 mEq/L, chloride 98 mEq/L, carbon dioxide 24 mEq/L, blood urea nitrogen 9 mg/dL, serum creatinine 0.6 mg/dL, glucose 119 mg/dL, calcium 8.6 mg/dL, albumin 3.9 mg/dL, phosphorus 2.5 mg/dL, and magnesium 2.1 mg/dL. Liver function tests were within normal limits, with the exception of lactate dehydrogenase that was elevated at 728 IU/L. Prothrombin time (PT) was 16.5 sec (normal 12.7―15 sec), international normalized ratio (INR) 1.24, and activated PTT (aPTT) was 38.3 sec (normal 24.7―35.9 sec). D-dimer was >20 μg/mL (normal 0―0.4 μg/dL) and fibrinogen was 424 mg/dL. A complete blood count (CBC) showed a white blood cell (WBC) count of 11,400/μL, hemoglobin of 11.6 g/dL, and platelet count of 14,000/μL. Review of the peripheral smear revealed the presence of anisocytosis and ovalocytes, and the absence of fragmented red blood cells (schistocytes). The platelet count had dropped from 316,000/μL to 14,000/μL in a 7-day period (see Fig. 1). Home medications included zolpidem, lorazepam, duloxetine, famotidine, fentanyl, hydromorphone, and ondansetron. Doppler ultrasound study showed a completely occlusive thrombus within the left internal jugular vein extending to the subclavian, axillary, brachial, and basilic veins. The patient had recently had a left subclavian central venous catheter (CVC) placed 11 days prior to presenting to the EC. The CVC was removed, but no anticoagulation was started secondary to severe thrombocytopenia.