Serum ferritin in patients with various haemolytic disorders.

174 serum ferritin assays in 121 patients with various haemolytic disorders have been performed. The mean serum ferritin levels were significantly increased in all these disorders in contrast to healthy controls. The highest serum ferritin levels were found in pyruvate kinase (PK) deficiency, moderate increase was observed in hereditary sphaerocytosis (HS) and in autoimmune haemolytic anaemia (AIHA) with massive haemolysis and in glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. Mild elevation of serum ferritin levels was depicted in paroxysmal nocturnal haemoglobinuria (PNH), in beta thalassaemia minor and in other types of haemoglobinopathies. The range of values was associated with a degree of haemolysis and its relation to duration of the disease was not apparent in most cases. Highly significant differences between serum ferritin levels in splenectomized and non-splenectomized patients with HS and between serum ferritin levels in patients with AIHA with massive haemolysis or in remission were found. As compared to normal controls, significant increase of serum ferritin levels was observed even in patients with AIHA in remission or in splenectomized patients with HS. In two patients with PK deficiency the levels exceeding 2,000 micrograms/l indicated manifest iron overload. A reliability of serum ferritin assay as an index of iron stores in haemolytic disorders has been discussed.     

Mechanism of haemolysis by Vibrio vulnificus haemolysin

The haemolytic action of Vibrio vulnificus haemolysin (VVH) was compared to that of streptolysin O (SLO). Both were cholesterol-binding haemolysins, but differed in the release of haemoglobin (Hb). In the first step of haemolysis, the haemolysins were temperature-independently bound to the cholesterol site on the target erythrocyte membrane. This was followed by the rapid release of K+, which is an intra-erythrocyte marker. Hb was then released, in different ways. In the case of VVH, Hb was released slowly after a relatively long lag, whereas with SLO, Hb was released as rapidly as K+. Haemolysis by VVH was inhibited by the addition of 30 mM-dextran 4 (mean Mr 4000), which is considered to be an effective colloid-osmotic protectant. The results therefore indicated that haemolysis by VVH (like that by Escherichia coli alpha-haemolysin and Staphylococcus aureus alpha-toxin) was caused by a colloid-osmotic mechanism. Both K+ and Hb release caused by VVH proceeded temperature-dependently, and the membrane fluidity of liposomes prepared with lipids extracted from sheep red blood cell membranes increased above 20 degrees C. These results suggest that the temperature-dependence of the haemolysis by VVH is due to the requirement for an increase in the membrane fluidity during the formation of a transmembrane pore.     

Effect of an interaction between haematocrit and cryoprotectant concentration on freeze-thaw haemolysis of bovine erythrocytes.

The effect of the haematocrit and cryoprotectant concentration on freeze-thaw haemolysis of bovine red cells was studied. Two-milliliter samples of bovine blood with an haematocrit of either 20 or 60% were diluted with 2 ml of either 5, 4, 3, 2, or 1 m glycerol or DMSO in PBS or with PBS alone. The degree of haemolysis after freezing to ?79 °C and thawing was least in blood diluted with 4m cryoprotectant. At the lower concentrations of cryoprotectant, haemolysis was greater in blood with the higher haematocrit, but this effect decreased as the cryoprotectant concentration was increased and was negligible at the optimal concentration.     

Fibrinogen-Fibrin Degradation Product Levels in Different Types of Intravascular Haemolysis

To examine the possibility that intravascular haemolysis may lead to intravascular coagulation we have compared the degree of fibrin deposition, as measured by levels of serum fibrinogen-fibrin degradation products (F.D.P.), in two different types of intravascular haemolysis associated with red cell fragmentation. F.D.P. levels in 56 patients with intravascular haemolysis secondary to prosthetic heart valves were compared with those in 18 patients who had microangiopathic haemolytic anaemia (M.H.A.) associated with malignant hypertension or renal disease. F.D.P. levels were raised in almost all the patients with M.H.A., and this group had significantly higher levels than any of the valve replacement groups. In contrast, in the prosthetic valve patients F.D.P. levels were usually normal and bore no relation to the degree of haemolysis. It is suggested that in the absence of other precipitating factors intravascular haemolysis will not initiate intravascular coagulation. In M.H.A., while the intravascular haemolysis appears to be a consequence of an underlying intravascular coagulation, it is likely that persistence of the coagulation disturbance is related more to factors such as small vessel damage than to the release of any thromboplastic substances from fragmented red cells.     

Antibodies to actin in autoimmune haemolytic anaemia

Background  

In autoimmune haemolytic anaemia (AIHA), autoreactive antibodies directed against red blood cells are up-regulated, leading to erythrocyte death. Mycoplasma suis infections in pigs induce AIHA of both the warm and cold types. The aim of this study was to identify the target autoantigens of warm autoreactive IgG antibodies. Sera from experimentally M. suis-infected pigs were screened for autoreactivity.     

Haemolysis during sample preparation alters microRNA content of plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.     

Heat-labile and heat-stable haemolysins of Campylobacter jejuni

Abstract During studies on the virulence mechanisms of Campylobacter jejuni clinical isolates it became apparent that some strains produced one or more haemolysins and some did not. There was no great difference between Group C (cholera-like) strains and Group D (dysentery-like) strains. The protein haemolysin(s) showed a spectrum of activity against erythrocytes from different animals; with maximum activity against rabbit and minimal activity against chicken erythrocytes. The results suggested a two-stage activation mechanism for haemolysis which involved a multi-hit lytic activity. It was concluded that the C. jejuni haemolysins were not identical to those described in other organisms and they may be involved in iron acquisition in vivo.     

Glucose-dependent Protection by Dietary Selenium against Haemolysis of Rat Erythrocytes <Emphasis Type="Italic">in vitro</Emphasis>

THE occurrence in man of drug-induced haemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes¹ suggested the possibility of an analogy to the haemolysis which occurs in vitamin E deficient red blood cells. Cohen and Hochstein² have shown that haemolysis in G6PD deficient cells is associated with the inability of the cell to generate adequate reduced glutathione (GSH) through GSSG reductase because of the impaired generation of NADPH. Moreover, there is evidence that glucose protects red blood cells from haemolysis by its ability to provide NADPH through G6PD which subsequently generates GSH³. The G6PD deficient cell, however, cannot maintain an adequate concentration of GSH in the cell, even in the presence of glucose⁴, whereas the normal cell can maintain a normal concentration of GSH in the presence of glucose, preserving the integrity of the red blood cell. Vitamin E protects red blood cells from haemolysis whether supplied in vivo or in vitro and its effect has usually been demonstrated without glucose in the incubation medium. Although selenium prevents many of the same deficiency symptoms as vitamin E, it has not been uniformly effective in preventing the in vitro haemolysis of red blood cells. If a protective action of selenium against haemolysis were dependent on the presence of GSH, or if selenium were involved in the generation of GSH, selenium would not be expected to prevent haemolysis unless glucose was present in the incubation medium to provide a constant source of NADPH for the generation of GSH from GSSG through GSSG reductase (Fig. 1).     

Haemolysis complicating viral hepatitis in patients with glucose-6-phosphate dehydrogenase deficiency.

Out of 20 patients with viral hepatitis whose glucose-6-phosphate dehydrogenase (G-6-PD) levels were normal, 14 had clinical evidence of a mild to moderate degree of haemolysis but in all the patients studied the half life of chromium-51-labelled red cells was shortened. Out of 18 viral hepatitis patients deficient in G-6-PD 17 had clinical evidence of haemolysis, and in eight this was more severe than in the group with normal G-6-PD values. Massive intravascular haemolysis occurred in four, three of whom died. The massive haemolysis was attributed to the presence of additional drug-induced oxidative stress to the G-6-PD-deficient red cells.     

Lysis of red blood cells in some haemolytic anaemias--the lytic effect of agkistrodon piscivorus venom in vitro.

The lysis of red blood cells induced by the Agkistrodon piscivorus venom (APV) in vitro in the presence and/or in the absence of plasma was examined in autoimmune haemolytic anaemia (AIHA), hereditary spherocytosis (HS) and paroxysmal nocturnal haemoglobinuria (PNH). Virtually haemolysis did not differ from that of normals in AIHA, it was slightly increased in HS and significantly higher in PNH, however, only in the presence of autologous or normal homologous plasma. The mechanism of PNH blood cells lysis which is probably related to the activation of the third complement component is discussed.     

Effects of warm acclimation on serum osmolality,cortisol and hematocrit levels in the Antarctic fish, <Emphasis Type="Italic">Trematomus bernacchii</Emphasis>

Antarctic fish, such as the Trematomus bernacchii, living at −1.9°C maintain a serum osmolality of around 600 mOsm kg⁻¹, nearly twice that of temperate fish. Upon warm acclimation, Antarctic fish significantly lower their serum osmolality. It has been suggested that this response to warm acclimation is due to stress. The purpose of this study was to determine, whether upon warm acclimation there was a change in the levels of the stress hormone cortisol and hematocrit associated with the decrease in serum osmolality. T. bernacchii were warm acclimated up to 4 weeks and serum osmolality, cortisol and hematocrit were measured. Upon warm acclimation to +1.6 and +3.8°C over the course of 4 weeks, T. bernacchii significantly lowered their serum osmolality (from 547 ± 4 mOsm kg⁻¹ to 494 ± 6 and 489 ± 4 mOsm kg⁻¹, respectively), yet did not alter their serum cortisol (29 ± 6 nl ml⁻¹) or hematocrit (22 ± 1%) levels. These results suggest that warm acclimation does not induce a stress response in T. bernacchii.     

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

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

Production and novel quantification of haemolysins produced by coagulase-negative staphylococci isolated from subclinical mastitis in sheep

Coagulase-negative staphylococci producing cell-damaging toxins were isolated from the milk of sheep with subclinical mastitis. The haemolytic activity of Coagulase-negative staphylococcal strains was assessed on solid and liquid culture media. More than 61% and 76% of the tested strains on solid media produced evidence of alpha- and delta- haemolysins and more than 78% produced synergistic haemolysis. However almost all isolates producing haemolysin in liquid culture media produced only very few units of haemolysin compared to the positive control of five Coagulase-positive strains of staphylococci. It was concluded that solid media are better for classifying Coagulase-negative staphylococci as producers or not of haemolysins, and liquid media for measuring the size of this activity within the first few hours of intramammary infection.     

Comparison of oxidative stress markers in vaginal deliveries with or without epidural analgesia

Abstract

Background

It has been demonstrated that oxidative stress can induce red blood cell rigidity and haemolysis, which in turn can cause hyperviscosity and hyperbilirubinaemia, respectively. However, haemolysis may be associated with a low level of haemoglobin, which reduces whole blood viscosity (WBV). Bilirubin can behave as antioxidant or oxidant, and one uncharted course for diagnostic pathology is how or whether bilirubinaemia and viscosity are associated. Further, oxidative stress is now being assessed using lipoprotein-a (Lp(a)), among other things but whether it is associated with blood viscosity has not been established.

Aim

This study investigates the association and correlation of haemoglobin level and WBV with serum Lp(a) and bilirubin levels in a general population of patients.

Materials and methods

Sixty-eight cases that were tested for Lp(a), concomitantly with full blood count and liver function, in our archived clinical pathology database were used in this study. WBV levels were determined using a validated formula. Multivariate and univariate analyses as well as correlation were performed.

Results

WBV was found to be significantly associated with bilirubin (P < 0.02), but not with Lp(a). Haemoglobin concentration was inversely correlated with Lp(a) (P < 0.04), but not with bilirubinaemia.

Conclusion

This pilot study suggests that hyperbilirubinaemia and hyperviscosity are associated and positively correlated. Consideration of whether serum bilirubin (as an indirect index of oxidative stress) can be used in combination with WBV (as index of macrovascular effect of oxidative stress) to assess oxidative damage is recommended.     

Effect of vitamin C and zinc on osmotic fragility and lipid peroxidation in zinc-deficient haemodialysis patients

Peroxidation of the membrane lipid structure of red blood cell leads to haemolysis and anaemia in haemodialysis patients. Dietary constituents of antioxidant vitamins and trace elements may play an important role in protecting against oxidant damage. In this study, the effects of supplementation of vitamin C and zinc on osmotic fragility and lipid peroxidation of erythrocytes were investigated in 34 zinc-deficient haemodialysis patients. Sixteen sex- and age-matched normal volunteers acted as controls. Patients were randomized to receive vitamin C (250 mg day(-1)), zinc (20 mg day(-1)) or a placebo treatment for 3 months. The levels of vitamin C, zinc, malondialdehyde (MDA) and osmotic fragility were measured initially and 3 months after supplementation. Mean serum concentration of vitamin C and zinc increased significantly in the groups at the end of the respective study periods. Supplementation with vitamin C and zinc improved osmotic fragility, and decreased the level of MDA in the groups, but some side-effects (i.e. nausea, vomiting, fever, muscle pain, weakness) were observed during the zinc treatment. The results showed that the supplementation of both treatments decreased osmotic fragilty and MDA in zinc-deficient haemodialysis patients. However, vitamin C treatment was found to be safer than zinc supplementation.     

免疫反应及相关细胞因子在EV71 相关手足口病 合并肺水肿中的作用

目的:探讨细胞、体液免疫应答以及相关的细胞因子在EV71相关手足口病合并肺水肿中的作用。方法:将90例经鉴定为EV71感染患儿分为手足口病合并肺水肿组38例,手足口病无并发症组52例,并设查体健康对照组28例。ELISA法检测90例EV71感染引起的手足口病患儿血清IL-6、IL-10、TNF-α及IFN-γ含量;采用流式细胞仪对血液中CD3+T、CD4+及CD8+T细胞百分比进行检测;采用免疫比浊法对90例EV71感染引起的手足口病患儿血清免疫球蛋白(IgG、IgA及IgM)及补体C3、C4含量进行检测。结果:手足口病合并肺水肿组患儿血清IL-6、IL-10及IFN-γ含量明显升高,同时伴随CD4+及CD8+T细胞百分比下降;手足口病合并肺水肿组,无并发症组及健康对照组患儿血清IgM分别为1.85±0.73,1.46±0.790和0.88±0.39,三组之间差别具有统计学意义(F=14.967,P<0.05)。而IgG与IgA在三组之间无明显变化(X2=5.535,P>0.05;F=1.988,P>0.05);手足口病患儿血清C3及C4含量均明显低于健康对照组(F=46.079;62.794,P<0.05)。结论:由IL-10及IFN-γ的异常释放而引起的广泛的中枢及外周神经系统炎症反应和T淋巴细胞衰竭是引起EV71合并肺水肿病程进展的重要原因;在EV71感染后引发的手足口病进程中存在IgM的大量释放,且伴随补体C3、C4的消耗。     

Monocyte receptors for the Fc portion of IgG increase in number in autoimmune hemolytic anemia and other hemolytic states and are decreased by glucocorticoid therapy

Peripheral blood monocyte receptors for the Fc portion of IgG were quantitatively studied in 43 normal subjects, in 14 patients with warm antibody autoimmune hemolysis (AIHA), and in nine individuals with nonantibody-mediated hemolysis. Monocytes of normal females expressed significantly greater numbers of Fc gamma receptors than did similar cells from male subjects, with no difference in affinity for the IgG1 probe. Monocyte Fc gamma receptor number was increased in patients of both sexes with AIHA; a similar, but smaller, increase in monocyte Fc gamma receptor number was noted in patients with nonantibody-mediated hemolysis. Glucocorticoid administration was associated with a dose-dependent decrease in monocyte Fc gamma receptor number in normal volunteers and patients. Possible etiologic mechanisms and pathogenetic consequences of enhanced monocyte Fc gamma binding in AIHA are discussed.     

Improved preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM)

Various fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR-LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OsO₄ and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing. © 1993 Wiley-Liss, Inc.     

系统性红斑狼疮患者血清MBL水平变化及其临床意义

目的：研究系统性红斑狼疮（SLE）患者外周血中甘露糖结合凝集素（MBL）水平与SLE的临床表现、经典的血清学指标及疾病活动性指数的相关性。方法：应用固相酶联免疫吸附（ELISA）法检测54例（其中活动期39例、缓解期15例）SLE患者和45名健康志愿者外周血MBL水平,同时检测SLE患者外周血抗dsDNA抗体、C3补体、白细胞计数及循环免疫复合物（CICs）水平,并记录SLE患者主要临床症状（肾脏系统受累、神经系统受累、严重感染等）,疾病活动度用SLEDAI记分。结果：系统性红斑狼疮患者外周血MBL水平显著低于健康志愿者（P〈0．01）;特别是活动期SLE患者血清MBL水平明显降低,与缓解组比较差别具有统计学意义（P〈0．01）;SLE患者血清MBL水平与SLEDAI、抗dsDNA抗体、CICs水平呈负相关,与白细胞计数及C3补体水平呈正相关;并发严重感染的SLE患者组血清MBL水平显著低于无受累组（P〈0．01）。结论：SLE患者血清MBL水平明显降低,血清MBL水平与SLEDAI、抗dsDNA抗体、C3水平、CIC水平、白细胞水平之间明显相关,血清MBL水平较低的患者更易发生比较严重的感染,血清MBL水平可能是SLE潜在生物学标记。     

Autoimmune haemolysis: an 18-year study of 865 cases referred to a regional transfusion centre.

Clinical and serological records of 865 patients with confirmed autoimmune haemolysis (AIH)--a much larger series than any previously reported--were critically reviewed nd analysed. A proposed new classification for AIH based on serological findings differs from traditional classifications in that a new category of "mixed" AIH has been defined in which both "warm" and "cold" autoantibodies are present, and both are capable of causing haemolysis. Patients in this mixed group tend to have severe disease that may run a chronic intermittent course. The presentation of cold agglutinin disease is much more variable than has been seen in previous studies, haemolysis due to low titre autoantibodies being common. The AIH associated with pregnancy, usually considered as being of bad prognosis, is often mild and self limiting in the absence of other associated disorders.