Abnormal Hemolysis Research Articles

A Systematic Literature Review of Clinical and Economic Outcomes for Patients with Paroxysmal Nocturnal Hemoglobinuria in Clinical Trials and Real-World Settings

Purpose: The purpose of this study was to systematically identify and evaluate published literature reporting clinical and economic outcomes associated with paroxysmal nocturnal hemoglobinuria (PNH) across clinical trials and real-world settings. Background: PNH is a rare, acquired hematopoietic stem cell disorder characterized by abnormal hemolysis of red blood cells.1,2 This complement-mediated hemolysis results in low hemoglobin [Hb] and high lactate dehydrogenase [LDH] levels, as well as symptoms including fatigue, dyspnea, pain, and hemoglobinuria.1-5 Patients frequently require costly hospitalizations and transfusions.4-10 Various measures of clinical response are used to assess treatment effectiveness and prognosis for patients with PNH, including laboratory values and transfusion dependence, but they are not consistently reported. This systematic literature review (SLR) describes clinical and economic outcomes reported in clinical trials and real-world/observational studies. Methods: Publications were collected through systematic searches for English language studies in MEDLINE, Embase, and Cochrane libraries with no date restriction, supplemented by abstracts and associated posters from recent major conferences (2020-2021). Studies were screened for relevance by two researchers independently, with disagreements adjudicated by a third reviewer. Studies were screened at 2 levels (title/abstract and full-text) using pre-specified criteria. Clinical trials and observational studies reporting clinical response (transfusion frequency, Hb levels, LDH levels, or reticulocyte counts), healthcare resource utilization, or costs were included in this descriptive analysis. This SLR is registered with PROSPERO (CRD42022314640). Results: Among 1607 unique publications following de-duplication, 105 studies with samples of at least 25 patients reported clinical response outcomes of interest. This included 29 clinical trials and 76 studies in real-world settings. Across these studies, methods of measuring clinical response varied: 12/29 clinical trials and 23/76 observational studies reported mean or median Hb during follow-up. These values were 11/29 and 15/76 for mean or median LDH and 4/29 and 6/76 for mean or median reticulocyte count, respectively. Mean Hb levels and mean LDH levels were the most commonly reported measures. Mean Hb levels at baseline ranged from 6.8-11.1 g/dL for patients in clinical trials and from 8.0-11.4 g/dL for patients in observational studies. Mean LDH levels ranged from 228-2293.3 U/L for patients in clinical trials and from 518-3880 U/L for patients in real-world studies. During study follow-up, mean Hb levels ranged from 7.6-12.8 g/dL and 6.8-11.9 g/dL for patients in clinical trials and real-world studies, respectively. Mean LDH levels during study follow-up periods ranged from 189-2419 U/L and 246.29-1555.45 U/L for patients in clinical trials and real-world studies, respectively. At study completion, mean and median Hb levels were <12.3 g/dL in all studies reporting values (n=35), with the exception of 1 clinical trial. Similarly, mean and median LDH levels were >225 U/L in 25/26 studies. Reticulocyte counts during study follow-up were only reported in 10 studies. Limited data were available describing the economic burden associated with PNH. Only 3 studies reported direct costs, with total direct costs ranging from $88,251-$624,911 and PNH-related costs estimated to be $13,220. The proportion of patients who required at least 1 transfusion remained high, ranging from 4-100%. Discussion and Conclusions: The Severe Aplastic Anemia Working Party of the European Society for Blood and Marrow Transplantation recently proposed clinical response classification based on Hb level (for complete response: ≥12 g/dL), LDH level (≤1.5 x the upper limit of normal), absolute reticulocyte count (≤150,000/µL), and transfusion requirements (none).11 This SLR has identified considerable gaps and inconsistencies in the reporting of these outcomes. Available data reflect persistently low Hb levels and high LDH levels across both clinical trial and real-world study publications, with outcomes in real-world studies suggesting most patients have at best partial response to available treatment. These laboratory values, along with continued transfusion dependence, underscore the sustained clinical and economic burden of PNH. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

L-Glutamine and Crizanlizumab for Adults with Sickle Cell Disease (SCD) in Qatar: A Cost Effectiveness Analysis

Background :Sickle cell disease (SCD) is a hereditary disease that is caused by autosomal recessive gene fault in the beta (β) allele of the hemoglobin (Hb) gene. As a result, sickled cells are characterized by easy and abnormal hemolysis with resultant varying degrees of anemia. Globally, the incidence of SCD is estimated to reach 400,000 persons per year, and in the United States alone, for example, the prevalence estimation is approximately 100,000 patients. Possible clinical presentations of SCD may come from different pathophysiologic mechanisms: the disfiguration of the RBC with subsequent loss of function can lead to vascular occlusion and a short lifetime of these RBCs that leads to hemolysis. The most severe and serious manifestation of SCD is the recurrent acute pain, or better known as vaso-occlusive crisis (VOC). Additionally, other clinical manifestations that SCD patients may show are acute complications such as acute chest syndrome (ACS), recurrent infections, kidney necrosis, and stroke. Such complications may affect multiple organs and can result in early death. Acute pain crisis is another common complication of SCD and is usually managed with pain medications, especially opioids,This is the first study to address two of novel therapies in patients with SCD in the Middle East. Our study was comprehensive in terms of outcome mostly encountered by SCD patients which is VOC and inclusivity of interventions mostly used for its management and was focused on the target population of in one of areas of high prevalence of SCD in the world. Our analysis tracked the CHEERS guidelines and checklist for reporting. To be also complete, we used only RCT evidence in our analysis. Additionally, in considering VOC, we ensured only studies with a definition compatible with that of the principal Crizanlizumab study were analyzed.Objectives:Treatment options for preventing vaso-occlusive crises (VOC) among sickle cell disease (SCD) patients are on the rise, especially if hydroxyurea treatment has failed. This economic analysis is conducted to assess the comparative clinical effectiveness, safety and acquisition cost of L-glutamine and Crizanlizumab for older adolescent and adults (≥16 years old) SCD in Qatar, with an emphasis on treatment costs and acute pain crises.Methods:We conduct a decision tree model, where we compare the clinical and economic outcomes of two novel FDA-approved medications which are available in Qatar; L-glutamine and Crizanlizumab over a time horizon of one year in a hypothetical cohort of adult SCD patients from a Qatar healthcare perspective. The main outcome is incremental cost per SCD-related acute pain crises averted. Model clinical parameters were derived from individual drug randomized trials, published literature, whereas cost parameters from Qatar healthcare payer system. A sensitivity analysis was carried out, and the study results were robust around model inputs. Costs were converted to 2020 US dollars.Results:Study results showed that both treatment modalities' costs were the main driver of this analysis, with average annual cost of the treatments per patient being $189,014 for Crizanlizumab (5mg/Kg), $143,798 Crizanlizumab (2.5mg/Kg) and $74,323 for L-glutamine. The probability of no first time SCD-related VOC averted were 0.001/year for Glutamine, 0.26/year for Crizanlizumab (5mg/Kg) and 0.34/year for Crizanlizumab (2.5mg/Kg). Lower dose Crizanlizumab (2.5mg/Kg) dominated the higher one (5mg/Kg). The ICER of Crizanlizumab (2.5mg/Kg), when compared to L-Glutamine was $81,265 per SCD-related VOC averted. When comparing Crizanlizumab (5mg/Kg) and L-Glutamine, Crizanlizumab (5mg/Kg) showed higher efficacy, yet the Crizanlizumab ICER was at $459,620 than L-glutamine.Conclusions:Crizanlizumab (2.5mg/Kg) may be cost-effective interventions yet it is not the approved dose for preventing VOC in adolescents and adults with sickle cell disease. Crizanlizumab (5mg/Kg) was more cost effective than the approved L-glutamine per SCD vaso-occlusive crisis prevented. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effect of percutaneous paravalvular leak closure on hemolysis.

To study the effect of percutaneous paravalvular leak closure on hemolysis. Although transcatheter PVL closure reduces heart failure and mortality in symptomatic patients with paravalvular leaks (PVL), little is known about its effect on hemolysis. We retrospectively analyzed patients undergoing transcatheter mitral or aortic PVL closure (January 2005-December 2016) at Mayo Clinic. Patients with anemia or abnormal hemolysis markers (LDH, haptoglobin) were included in the analysis. The primary outcome was defined as hemoglobin increase ≥ 1.5 mg/dL, decrease in LDH above median or improvement in haptoglobin. Univariate and multivariate binary logistic regression modeling were used to determine predictors of successful treatment of hemolysis. Final study population included 168 patients (130 [77%] mitral, 38 [23%] aortic PVL). Primary outcome occurred in 70 patients (42%). Hemoglobin increased by 1.74 ± 1.69 mg/dL in patients who reached primary outcome. 57/168 (34%) patients required blood transfusion prior to PVL closure compared to 35/168 (21%) postprocedure. The mean reduction in LDH was 403 U/L. Multivariate regression showed that patients with mechanical valves were more likely to have successful outcome (P = 0.044). Percutaneous PVL closure is associated with modest improvement in hemolysis markers, increase in hemoglobin levels and reduction in blood transfusion requirements. This benefit is most significant in patients with mechanical valves.

Use of Ventricular Assist Device Acoustical Signatures to Detect Device Thrombosis

Background Pump thrombosis (PT), the most common cause of failure in ventricular assist devices (VADs), can be mitigated with earlier diagnosis before triggering any clinical events or requiring VAD replacement. Hypothesis Acoustical signatures of VADs can provide substantial information regarding PT. Methods We included 10 patients (6 axial, 4 centrifugal VADs) presenting with pump power elevations and recurrent abnormal hemolysis markers consistent with suspected PT. VAD sounds were recorded multiple times using a digital stethoscope (EKO Core) at baseline or before developing thrombosis (normal baseline), during thrombosis episodes and after treatment with heparin or tissue plasminogen activator (Fig. (a)). The 2 types of pumps were analyzed separately (42 recordings from axial and 30 recordings from centrifugal) using 3 steps: (1) Extracting features (time-frequency, acoustic and nonlinearity features) from each recording; (2) Feature importance ranking using Extreme Gradient Boosting (XGBoost) to evaluate which features were most relevant for distinguishing among different states; (3) Principal Component Analysis (PCA) for dimensionality reduction and 2D visualization (Fig. (b)). Results We found that separation among the different states is achieved using the combination of XGBoost feature importance ranking and PCA (Fig. (c)). The suggested pipeline of analysis can overcome the problems emerging from inter-subject variability. In both pumps, sounds recorded from VADs with PT had different signatures than baseline sounds. Better separation was observed for axial pumps relative to centrifugal pumps, which may be due to the difference in operating speeds (average 9400 vs 2600 rpms, respectively). We observed a slight overlap between the post treatment and baseline episodes, suggesting the possible presence of residual PT that is not identified by hemolysis biomarkers and/or pump powers. Conclusion Applying machine learning algorithms on pump acoustical signals provides substantial information regarding thrombosis states, and potentially allows for sensitive detection of PT assisting hemolysis biomarkers and pump powers.

Hemolytic assay for Huangqi Injection

ObjectiveHuangqi Injection is a preparation with an extract of Astragali Radix which has a long history of being used as a tonic to strengthen the body's immunity. Anaphylaxis and hemolysis are two main adverse drug reaction (ADR) of injections. Our study was aimed to establish an approach for the (ADR) prediagnosis of Huangqi Injection. MethodsAn in vitro model for anaphylactoid assay of Huangqi Injection based on the release rate of histamine and β-hexosaminidase of RBL-2H3 cells induced by injections and a colorimetric method based on the detection of hemoglobin resulted in the erythrocyte hemolysis for prediagnostic assaying the hemolytic ADR of injections were established. ResultsBoth histamine and β-hexosaminidase are the anaphylactiod mediators, but β-hexosaminidase release induced by Huangqi Injection could not be determined by spectrophotometry due to the interference of the injection itself. In addition, normal hemolysis and abnormal hemolysis were discovered during the experiment. The fingerprints and tannins in different batches of injections showed obvious differences, indicating that the content of tannins was related to abnormal hemolysis and higher histamine-secreting from RBL-2H3 cells. ConclusionThe results indicate that the hemolytic assaying method is not only suitable for prediagnostic assaying of hemolytic ADR of herbal medicine injection, but also partly reflects the anaphylaxis of herbal injections, and tannins may be the major factors causing abnormal hemolysis.

Iron Overload Effects On Immune System Through The Cytokine Secretion By Macrophage

Iron is essential for almost all organisms. However, free iron can be cytotoxic at high concentration and iron excess can have adverse effects on variety of cells, tissue, and organ functions. In immune systems, many reports have shown the effects of iron, some of which are complex and controversial. Iron deficiency has been reported to be associated with increased susceptibility to infection, but iron overload caused by dietary excess abnormal hemolysis or inherited disorders is also associated with heightened susceptibility to infections. On the other hand, elevated ferritin levels, primarily related to RBC transfusions, have been reported to increase the risk of acute and chronic GVHD in patients received hematopoietic cell transplantation. Although this iron-related risk of GVHD may reflect the organ damage, such as liver, kidney, and pancreas, it also may be caused by imbalance in immune systems.To exaggerate the effects of iron overload on immune system, we established iron-loaded models in mice. First, as acute iron-loaded model, 10mg of iron dextran, which is even equal to 200U of RCC transfusion in human (define as 200U of iron dextran), was intraperitoneally injected into mice once a day for 18 days. In peripheral blood, T cell and B cell populations were decreased but monocyte/macrophage and neutrophil populations were increased as compared in control mice administered with dextran only. And notably, regulatory T cell (CD4+Foxp3+; Treg) population was significantly reduced in iron loaded mice than control mice (1.40% vs 0.58%). Next, as chronic iron-loaded model, 2U of iron dextran was injected once a week 45 times. Although significant difference was not observed in Treg population in PB, that in spleen was significantly high in iron-loaded mice as compared with that in control mice (3.2% vs 1.7%, p<0.05). From these data, we suspected that the excess of iron reduces Treg population in some organs, thereby effects on immune function.To clarify the mechanism of Treg-reduction by iron overload, we established an intermediate iron-loaded model, in which 50U of iron dextran was injected into mice once a week for 10 weeks, and following experiments were performed in this model. As previous examinations, Treg population in spleen was reduced in iron-loaded mice (0.86% vs 0.46%). T helper 17 (Th17) cells are recently described lymphocyte subset with common precursors with Treg but with opposing actions to Treg. The difference of Th17 cells could not be detected in spleen because of its too small population, the population of Th17 was increased in small intestine of iron-loaded mice (0.85% vs 1.51%). Treg and Th17 cells can be differentiated from naïve Th cells (CD4+CD62L+) with stimulation of specific cytokines (Treg; TGFβ, IFNγ, and IL4, Th17; TGFβ, IL-6, and IL-1β). We analyzed the direct effects of iron on Treg and Th17 cells during the in vitro differentiation from naïve T cells. Both in Treg and Th17 cell inductions, we could not detect significant differences between cells supplemented with iron dextran and those with dextran only. We also exaggerated the reactive oxide spesies (ROS) accumulated in induced T cell using RedCC1 staining. During the induction of Treg and Th17, although H2O2 supplement increased the ROS accumulation dose dependently, iron dextran-addition did not change the amount of ROS.Next, we analyzed the serum concentrations of cytokines in iron-loaded mice both in acute and intermediate models. IL-1β, and IL-23 levels were elevated in iron-loaded mice. These data suggested that iron overload reduces Treg cell population and increases Th17 cell population not directly, but through the cytokine secretion from environmental cells. So we evaluated the induction rates of Treg and Th17 cells from naïve T cells under co-culture with monocyte/macrophage. In this condition, Treg induction rate was significantly lower in iron dextran-supplemented cells (48.1% vs 35.3%), and Th17 induction was increased (8.4% vs 18.0%). Furthermore, mRNA expressions of IL-23 and IL-β in macrophages were increased with iron dextran-supplement in in vitro culture.These data suggest that iron overload changes the balance of Treg and Th17 cells through the proliferation and activation of macrophages, and thereby effects on the immunological condition of some disease, such as GVHD and autoimmune. Disclosures:No relevant conflicts of interest to declare.

The Anaphylactoid Constituents in Xue-Sai-Tong Injection

Xue-Sai-Tong injection, a traditional Chinese medicine with total saponins of Sanqi ginseng as active ingredients, has been used for more than 500 years to treat coronary artery disease in China. Anaphylactoid reaction induced by Xue-Sai-Tong injection was one of the main adverse drug reactions which has occurred frequently in recent years. It is of importance to elucidate its anaphylactoid constituents. The in vivo anaphyalctoid tests indicated that the anaphylactoid mediators could be used as indexes to evaluate the anaphylactoid action. Further, the in vitro model based on determining the mediators release from the degranulation of mast cells and RBL-2H3 cells stimulated by Xue-Sai-Tong injection was explored. Mediators released from mast cells and RBL-2H3 cells caused by Xue-Sai-Tong injection were determined by comparison of the methods of fluorospectrophotometry, ELISA, and spectrophotometry, respectively, revealing that the histamine release induced by the Xue-Sai-Tong injection could not be assayed accurately by the method of fluorospectrophotometry because of the interference of saponins and unknown components in the injection. The rat peritoneal mast cell was also not an optimal cell model for determining histamine and β-hexosaminidase release due to the higher spontaneous release ratio during the cell collection. Thus, ELISA determination of the histamine release from RBL-2H3 cells is a suitable in vitro model to assay the anaphylactoid reaction of Xue-Sai-Tong injection. Previously, abnormal hemolysis in some batches of Xue-Sai-Tong injection was observed in the course of their HD₅₀ (half hemolytic dosage) determination. This study further found that injections which exhibited an abnormal hemolysis phenomenon also caused a higher release of the anaphylactoid mediators from RBL-2H3 cells, indicating the HD₅₀ could be an auxiliary index to evaluate anaphylactoid action of the herbal injection indirectly. Research for anaphylactoid components in Xue-Sai-Tong injection indicated that proteins with over 10 KDa of molecular weight, but not ginsenosides, could be the main constituents inducing the release of anaphylactoid mediators from RBL-2H3 cells. An HPLC method for protein determination in the Xue-Sai-Tong injection was established subsequently, and the content of proteins with molecular weights of over 10 KDa in the injections showed an obviously positive correlation with the histamine release induced by the injections. In addition, taking ginsenoside-Rd coupled with BSA as an example, the hapten property of ginsenosides was studied and the ratio of ginsenoside-Rd to BSA was determined to be 8:1 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the ginsenoside-BSA conjugate showed a stronger action to stimulate histamine release from the RBL-2H3 cells.

Prediagnostic methods for the hemolysis of herbal medicine injection

Ethnopharmacological relevanceThe Xue-Sai-Tong injection, a traditional Chinese medicine injection with total saponins extracted from Sanchi Ginseng, has been used for more than half a hundred years to treat coronary artery disease. The study is to establish a prediagnostic method for the hemolytic adverse effect of herbal medicine injection by taking Xue-Sai-Tong injection as an example. Materials and methodsA new method named “fuzzy dissemination” was established to identify the hemolytic ginsenosides in Xue-Sai-Tong injection on the basis of fuzzy changes of individual ginsenosides in the injections altered by re-adding the fractions prepared from the total saponins and statistic analysis between hemolytic degrees and individual ginsenosides. Related substances test, safety tests and fingerprints of the injections in different batches were tested. ResultsHD50, P50 and interactions on hemolysis of individual ginsenosides were examined. Experiment indicated that the content of Rg1, Rg2, M51 (an unknown ingredient with retention time at 51min in HPLC) and M70 in Xue-Sai-Tong injection showed a significant positive correlation with hemolytic degree, and the content of R1, Re, Rb1 and Rd showed a significant negative correlation with hemolytic activity. Furthermore HD50 of injection exhibits superiority to other tests for the hemolysis of injections. Abnormal hemolysis in some batches of injections was observed, but there were no significant differences among injections of different batches in related substances test, safety test and fingerprints. ConclusionsThis is an original method to analyze active ingredients of a complicated integrity instead of studying on individual ingredients, it showed that the interactions of some individual ginsenosides and some unknown micro-ingredients in Xue-Sai-Tong injection were the major factors causing hemolysis, and this method could also be utilized in research of corresponding aspects. HD50 of injection can reflect the changes of hemolytic property of injections caused by not only the change of active constituents of injection, but also the auxiliary materials. Thus it was recommended as an index for the hemolytic prediagnosis of the injections in practice.

Treating Dense Deposit Disease by controlling the C3 convertase

In DDD, fluid-phase dysregulation of the AP of the complement cascade leads to deposition of complement activation products (iC3b, C5, C6, C7, C8, C9) along the GBM. Our data show that a soluble form of the transmembrane complement regulator sCR1 prevents abnormal hemolysis in vitro in presence C3Nefs. In addition, its administration to DDD mouse models results in restoration of plasma C3 levels and reduction of C3 deposition along the GBM. Similar therapeutic effects have been seen using human or mouse purified factor H in DDD mouse models.4,5

Production of Nitrosoglutathione Through Met Hemoglobin Reactions with Nitrite and Low Molecular Weight Thiols

Acellular hemoglobin (Hb) in the circulation, due to abnormal hemolysis is generally considered to be a source of toxicity. The toxicity is exacerbated by the presence of inflammatory conditions such as endothelial dysfunction that result in decreased levels of NO and nitrite in the plasma. Under some conditions, Hbs can actually generate bioactive NO that can compensate for depressed levels of NO due to inflammation and NO-dioxygenase activity of acellular Hbs. In the present work, we present a systematic biophysical study of how R and T state forms of met Hb in the presence of nitrite and thiols can generate nitrosoglutathione (GSNO). GSNO, an efficient vasodilator, is known to be in equilibrium with a pool of thiol/S-nitrosothiol containing proteins in blood vessels. Thus nitrite and thiol mediated GSNO production by acellular Hbs can be viewed as a means both to generate long lived forms of bioactive NO in the circulation and to replenish depleted S-nitrosothiol levels in the vasculature.

Blood Sampling Through Peripheral Venous Catheters Is Reliable for Selected Basic Analytes in Children

The goal was to determine the interchangeability of peripheral venous catheter (PVC) and venipuncture blood sampling (BS). Paired blood samples from hospitalized children were obtained through venipuncture and from existing PVCs, following discard of 2 mL of blood. Comparisons of 9 complete blood count indices (white and red blood cell counts, hemoglobin and hematocrit levels, mean corpuscular volume, mean corpuscular hemoglobin level, red blood cell distribution width, platelet count, and mean platelet volume) and 5 basic chemical analysis indices (sodium, potassium, glucose, chloride, and urea levels) were performed, and hemolysis was documented. Irrespective of gauge, blood samples were obtained successfully from 40 (85.1%) of 47 PVCs, with no abnormal hemolysis. BS through venipuncture took longer than BS from PVCs (175.8 +/- 229.6 vs 104.5 +/- 53.4 seconds; P = .053) and was associated with significantly more distress/crying (73.1% vs 0%; P < .001). There were no significant differences between venipuncture and PVC samples (paired t test). Twenty-one (6%) of 348 pairs analyzed with the Clinical Laboratory Improvement Amendment standards fell outside the range of acceptable variance (8 of 21 aberrations were attributed to glucose measurements). Bland-Altman analysis indicated that, with the exclusion of glucose measurements, BS from PVCs is reliable, with 29 (6.5%) of 448 pairs exceeding the limits of agreement. Of those, 9 cases were clinically significant, but none would have altered clinical management. PVC sampling was shown to be a pain-reducing method that can be used for children for selected basic analytes. The findings for glucose were unreliable.

Bilirubin Toxicity to Human Erythrocytes: A More Sanguine View

In 1962 Watson1 observed that a small fraction of the bilirubin in blood from jaundiced newborns is associated with red cells. A few years later, Cheung et al2 showed that red cells undergo time- and concentration-dependent changes in morphology and metabolism, with eventual hemolysis, when incubated with bilirubin in buffer, particularly at concentrations of >340 μM (20 mg/dL). (In retrospect, it seems highly likely that the bilirubin solutions to which the cells were exposed were supersaturated and may have contained aggregated forms of bilirubin.3–7) The bilirubin-induced morphologic changes were illustrated by striking photographs similar to those published some 30 years later, without reference to the earlier work, by others.8,9 These changes were reversible by addition of serum albumin to the incubation medium and did not occur when albumin was present in the medium from the start. Subsequently, Brito et al10 have confirmed and extended these observations and recently reviewed the toxicity of bilirubin to red cells (again without alluding to the seminal earlier work). For the neonatologist, an important question is whether bilirubin-induced hemolysis is clinically significant in neonatal jaundice or in other unconjugated hyperbilirubinemias. The review implies that it is.10 There is, however, much evidence that it is not: 1. Gunn rats and patients with severe Crigler-Najjar syndrome have lifelong unconjugated hyperbilirubinemia yet no abnormal hemolysis. In fact, the human disorder was originally described as a “nonhemolytic jaundice,”11 and patients have been described who showed no abnormal erythrocyte morphology, reticulocytosis, or other evidence of hemolysis despite prolonged exposure to high plasma bilirubin concentrations.12–14 Comparisons between nonjaundiced and jaundiced rats with congenital unconjugated bilirubinemia (Gunn rats) showed no difference in hematocrit values in male rats and only slightly lowered values in jaundiced female rats.15 No difference … Address correspondence to Antony F. McDonagh, PhD, Division of Gastroenterology and the Liver Center, Room S-357, Box 0538, University of California, 513 Parnassus Ave, San Francisco, CA 94143-0538. E-mail: tony.mcdonagh{at}ucsf.edu

Red blood cell hemolysis during processing

Red blood cell (RBC) hemolysis has been reported in units of RBC for transfusion. This has significant clinical implications for transfused patients because the free hemoglobin dissociates into dimers that have to be bound to haptoglobin to be removed by the reticuloendothelial system. Once the binding capacity of haptoglobin has been exceeded, hemoglobinemia occurs. Hemolysis is caused by the breakdown of the RBC, causing release of hemoglobin and resulting in the discoloration of the plasma. Abnormal hemolysis in an individual RBC unit may be caused by several factors including inappropriate handling during processing of blood, inappropriate storage conditions, bacterial hemolysins, antibodies that cause complement lysis, defects in the RBC membrane, or an abnormality in the blood donor. The degree of hemolysis is described as the percent of free hemoglobin in relation to the total hemoglobin with appropriate correction for the hematocrit. The acceptable level of hemolysis has not been established in North America, but the value of 1% currently is used to assess biocompatibility of blood storage materials, whereas the Council of Europe has set the standard at 0.8%. This report emphasizes the need for the adequate control of the various processes that are involved in the preparation of RBCs from whole blood to minimize the occurrence of hemolysis. Careful evaluation of manufacturing processes will minimize RBC wastage caused by hemolysis.

Evaluation of a physiologic pulsatile pump system for neonate-infant cardiopulmonary bypass support.

An alternate physiologic pulsatile pump (PPP) system was designed and evaluated to produce sufficient pulsatility during neonate-infant open heart surgery. This hydraulically driven pump system has a unique "dual" pumping chamber mechanism. The first chamber is placed between the venous reservoir and oxygenator and the second chamber between the oxygenator and patient. Each chamber has two unidirectional tricuspid valves. Stroke volume (0.2-10 ml), upstroke rise time (10-350 msec), and pump rate (2-250 beats per minute [bpm]) can be adjusted independently to produce adequate pulsatility. This system has been tested in 3-kg piglets (n = 6), with a pump flow of 150 ml/kg/min, a pump rate of 150 bpm, and a pump ejection time of 110 msec. After initiation of cardiopulmonary bypass (CPB), all animals were subjected to 25 minutes of hypothermia to reduce the rectal temperatures to 18 degrees C, 60 minutes of deep hypothermic circulatory arrest (DHCA), then 10 minutes of cold perfusion with a full pump flow, and 40 minutes of rewarming. During CPB, mean arterial pressures were kept at less than 50 mm Hg. Mean extracorporeal circuit pressure (ECCP), the pressure drop of a 10 French aortic cannula, and the pulse pressure were 67+/-9, 21+/-6, and 16+/-2 mm Hg, respectively. All values are represented as mean+/-SD. No regurgitation or abnormal hemolysis has been detected during these experiments. The oxygenator had no damping effect on the quality of the pulsatility because of the dual chamber pumping mechanism. The ECCP was also significantly lower than any other known pulsatile system. We conclude that this system, with a 10 French aortic cannula and arterial filter, produces adequate pulsatility in 3 kg piglets.

Transgenic mouse models of sickle cell disease.

An array of sickle cell syndromes has been obtained in transgenic mice, expressing HbS or super HbS, from the asymptomatic phenotype similar to the human A/S state to a syndrome more severe than the human homozygous S/S state, inducing 100% fetal death. Anemia was observed in SAD and SAD (beta th/ beta +) neonates and disappeared during postnatal development. In adults, many features of sickle cell disease are found in transgenic mice, especially in SAD and SAD (beta th/ beta +) mice, including abnormal hemolysis, vasoocclusion, microthrombosis, infarct, priapism, chronic organ defects, and death on hypoxia. These mouse models are relevant to the study of the pathophysiology of sickle cell disease and the induction of vasoocclusion and to evaluate new therapeutic approaches in vivo. Clotrimazole and Mg2+ restore hydration of sickle cells and 12 C79 protected SAD mice from lethal acute hypoxia.

A STUDY OF THE TYPES OF BILIARY CALCULI IN GALLSTONE DISEASE OCCURRING AFTER HEART VALVE REPLACEMENT

To elucidate the cause of gallstone disease after heart valve replacement according to the types of gallstone, 140 patients with valvular heart disease undergoing valve replacement in the department from 1969 to 1990 were studied. Thirty-two of these patients, who had gallstones by abdominal echography, were evaluated for the nature, type and incidence of the calculi, as well as related biochemical findings including the liver function and occurrence of hemolysis, and relations to the presence or absence of hepatosplenomegaly. Pigment stones were echographically discovered in 19 of the 32 patients (59.4%) with gallstone disease after heart valve replacement, No relationship between the type of stone, position and type of valve replaced was observed. No association with gallstone disease and abnormal hemolysis or liver function was found. The high incidence of pigment gallstones complicated by hepatomegaly, however, suggests that gallstone disease after heart valve replacement may be caused by some liver disorders such as stasis of the liver.

Pulmonary excretion rate of carbon monoxide as an index of total bilirubin formation in adult male Wistar rats with common bile duct ligation.

Total bilirubin formation (TBF) in the rat after a short period of common bile duct ligation was studied by measuring the pulmonary excretion rate of CO (VECO). At postoperative day 3, the VECO of experimental animals was higher when compared with the preoperative VECO (p less than 0.005); whereas the VECO of control animals did not change. Also, on the 3rd postoperative day, the relative rate of early labeling of bilirubin following the administration of delta-aminolevulinic acid-5-14C, a preferential hepatic heme synthesis precursor, was similar between the experimental and control animals; only the experimental animals had an abnormal peroxide hemolysis test. We conclude that common bile duct ligation in the rat is associated with elevations in the VECO, indicating significant increases in TBF, and the source of the increase is probably of erythropoietic origin. This finding may be relevant to the understanding of the pathophysiology of obstructive jaundice in human neonates.

A New Familial Disorder with Abnormal Erythrocyte Morphology and Increased Permeability of the Erythrocytes to Sodium and Potassium

Extract: A newborn infant of Philippine parents was found to have a morphological abnormality of his erythrocytes consisting of an elliptical shape of the cells and one or more transverse slitlike areas of decreased density. These changes were also present in erythrocytes of the patient's father, a half-sister of the father, and four of the patient's six siblings. None of the affected family members had anemia or evidence of abnormal hemolysis, and erythrocyte survival by the radiochromium method was normal in three of the individuals studied. Erythrocytes from the affected family members had an increased degree of autohemolysis after incubation for 48 hr, but this was prevented almost entirely by addition of glucose. Glucose consumption in vitro by erythrocytes of the propositus occurred at a rate approximately 60% greater than that of normal controls. The intracellular sodium concentration of the erythrocytes was not different from that of erythrocytes from normal individuals, but a moderate decrease in intracellular potassium was found. When washed cells were incubated in a glucosefree medium, sodium gain and potassium loss were significantly greater than from cells of normal controls. When compared with normal values, efflux of radiosodium was increased during incubation of cells in glucose-containing medium. Erythrocytes from the affected individuals had decreased osmotic fragility, and osmometric measurements indicated a lesser degree of cell swelling in hypotonic solutions than occurred with cells from normal controls.Speculation: The findings in this family demonstrate that increased erythrocyte cation permeability need not be associated with an accelerated rate of hemolysis. In these individuals the absence of hemolytic disease may reflect a capacity of the erythrocytes to compensate for increased cation permeability by an elevated rate of glycolysis-linked ion transport. This compensatory process would appear to be virtually unaffected by interaction of the erythrocytes of these individuals with the spleen.

Hb Sogn (β14 Arginine)

Hb Sogn (β14 arginine) is a haemoglobin more unstable than Hb A, however, the individuals heterozygous for Hb Sogn present no symptom or sign of abnormal haemolysis, and in all probability they tolerate treatment with oxidant drugs like sulfonamides. Clinically, Hb Sogn is an innocuous haemoglobin variant.Hb Sogn could be demonstrated through three generations. There is no close genetic linkage between the locus for haemoglobin β‐chain and loci for Rh, Gc, and PGM1 characters.

Haemolysis and Erythropoiesis

Summary. Case records of patients with anaemia due to lack of iron, vitamin B12 or folate, or to abnormal haemolysis were reviewed with regard to magnitude of reticulocytosis and rate of haemoglobin rise after therapy. Greater absolute reticulocyte values were observed in hereditary and acquired haemolytic anaemias. Furthermore, and more significantly, the rate of haemoglobin rise in autoimmune haemolytic anaemia responding to corticosteroid administration was greater than in the deficiency anaemias during adequate therapy. Additional data are presented supporting the old concept that erythrocytic production is greater in haemolytic anaemias than in deficiency anaemias after specific therapy.