Prevalence of increased osmotic fragility of erythrocytes in German blood donors: Screening using a modified glycerol lysis test

Summary We screened for increased osmotic fragility of erythrocytes in 1464 healthy German blood donors. The osmotic fragility was determined by an acidified glycerol lysis test (AGLT) using glycerol-sodium phosphate-buffered NaCl solution. Since the original test described by Zanella et al. [23] showed only low specificity for hereditary spherocytosis, we used a modification with 0.0093M sodium phosphate-buffered glycerol-saline solution, pH 6.90, instead of the original 0.0053M sodium phosphate buffer, pH 6.85. Sixteen of the donors (1.1%) had a pathologic result, similar to that of 32 patients with hereditary spherocytosis: AGLT 50 <5 min (half-time of AGLT, defining normal and pathologic results). The osmotic fragility of the erythrocytes from 12 of these donors was further investigated using the conventional test with hypotonic NaCl solutions. With one exception, increased osmotic fragility was verified in all of them by both tests. Further hematologic data showed a mild reticulocytosis (2% and 2.6%) in two of the donors. One donor had a moderate reticulocytosis of 6.5%, probably due to a mild, previously undiagnosed spherocytosis; 99 of the donors had an intermediate result (AGLT 50: 5–30 min). Hypotonic lysis of their erythrocytes by the conventional method showed a normal result; there were no signs of increased hemolysis. Thus they are not definitely regarded as having increased osmotic fragility of their erythrocytes. Erythrocyte osmotic fragility shows a wide distribution range in the normal population and might be normally distributed. Thus the blood donors with pathologic AGLT (<5 min) probably represent only one end of a continuum of salt-dependent hemolysis, and not a separate entity. However, they did show additional minor signs of a functional defect of the erythrocyte membrane and therefore could be carriers of a spherocytosis trait. The frequency of carriers of an erythrocyte membrane defect (possible spherocytosis trait) could be as high as 1.1% in the general population and would distinctly exceed the prevalence of patients with apparent spherocytosis (0.02%).     

Recombinant factor VIIa reverses the anticoagulant effect of the long-acting pentasaccharide idraparinux in healthy volunteers

We investigated whether the anticoagulant effect of idraparinux, a selective long-acting factor Xa inhibitor, could be neutralized by recombinant factor VIIa (rFVIIa) in healthy male volunteers. We performed a randomized, placebo-controlled trial, comparing idraparinux [7.5 mg subcutaneous (s.c.)] followed at 3 h by rFVIIa [90 microg/kg intravenous (i.v.)] (n = 6), or idraparinux (7.5 mg s.c) followed after 1 week by rFVIIa (90 microg/kg i.v.)(n = 6). rFVIIa, given 3 h after idraparinux, significantly reversed the increased thrombin generation time (TGT), the increased activated partial thromboplastin time (aPTT) and prothrombin time (PT), and the reduced prothrombin fragment 1+2 (F1+2) levels caused by idraparinux, although no clear effect of rFVIIa on the endogenous thrombin potential (ETP) was observed. One week after idraparinux, injection of rFVIIa resulted in a similar relative reduction of the remaining increased aPTT, PT and TGT, with correction to pre-idraparinux values. A clear increase of F1+2 was observed, together with a small increase in ETP. We conclude that rFVIIa has significant effects on the idraparinux-inhibited thrombin generation and clotting parameters. These results suggest that rFVIIa may be useful in serious bleeding complications in idraparinux treated patients.     

The evolution of anticoagulant therapy

Arterial and venous thromboembolism are leading causes of morbidity and mortality around the world. For almost 70 years, heparins (unfractionated heparin and low molecular weight heparins) and vitamin K antagonists have been the leading therapeutic medical options for the treatment and prevention of thromboembolic disorders. Nevertheless, the many limitations of these traditional anticoagulants have fuelled the search for novel agents over the past 15 years, and a new class of oral anticoagulants that specifically target activated factor X and thrombin has been developed and is now commercially available. In this narrative review, the evolution of anticoagulant therapy is summarised, with a focus on newer oral anticoagulants.     

Combined effect of dextrose and sodium chloride on red cell osmotic fragility

Observations indicating an enhancing effect of sodium on hemolysis of red cells suspended in dextrose solution prompted study of the mechanism of this effect. Osmotic fragility of red cells from normal subjects and from patients was measured in 5% dextrose solution (5D/W), 5% dextrose combined with various concentrations of sodium chloride (5D/NaCl), lithium chloride, and potassium chloride. The influence of ouabain on osmotic hemolysis in 5D/ NaCl was also studied, and the glucose content of ghost red cells was determined when incubated in 5% dextrose with 0.05% NaCl (5D/O.05NaCl), as compared with 5D/W. In the presence of minimal amounts of NaCl (0.05%) at pH 4.8, average hemolysis was greater than in the absence of NaCl, ie, 39% vs 24% in normal subjects (P < 0.02) and 40% vs 31% in a group of unselected patients (P < 0.05). When the pH was adjusted to 7.4 in the patient group, the results were 34% vs 21% (P < 0.05). The sodium-induced enhancement of hemolysis in dextrose solution was virtually duplicated when LiCl and KCl were substituted for NaCl (P < 0.01 and < 0.05, respectively). On the other hand, in the presence of ouabain, the sodium-induced enhancement of hemolysis was abolished. The overall glucose content of ghost red cells incubated in 5D/0.05NaCl was 53% greater than in 5D/W (P < 0.005), whereas with ghost red cells depleted of adenosine triphosphate (ATP), it was only 32% greater (P < 0.01). These results suggest that glucose transport across the red cell membrane is enhanced by the sodium ion, presumably by triggering the membrane sodium-potassium ATPase system.     

Evaluation of single-tube osmotic fragility as a screening test for thalassemia

A single-tube osmotic fragility test has been proposed for thalassemia screening with a range of different concentrations of saline having been employed. We have compared the sensitivity and specificity of 0.32%, 0.34%, and 0.36% buffered saline, and on the basis of our findings, recommend the use of 0.36% saline. This gave definitely positive or equivocal results in 81 of 85 patients with beta thalassemia trait and in 4 of 4 with alpha(0) thalassemia trait. There were 14% false positive results in hematologically normal patients and 81% of the samples from patients with various variant hemoglobins gave positive results. The sensitivity was 95% and specificity 86%. The single-tube osmotic fragility test is potentially useful in under-resourced laboratories although it cannot replace automated red cell indices using electronic counters.     

Continuous flow method for determination of erythrocyte osmotic fragility

A simple and accurate micromethod for the determination of erythrocyte osmotic fragility is introduced. The method uses a laminar parabolic flow pattern, together with gravity, to retain cells in a long, small-diameter tube while a solution with decreasing osmolarity is passed through the tube. As the cells hemolyze, hemoglobin released from the cells is quickly removed by the axial flow pattern and monitored with a 547 nm optical detector for recording the hemolysis curve. Consequently, a continuous curve is obtained, with a peak occurring at the salt concentration that produces the maximum hemolysis rate. The advantages of this method are simplicity, accuracy, and small sample size (2 microliters of whole blood). The small sample size is of particular importance for infants. A comparison is made with the Parpart method using samples from 18 normal adults. Results are also given for a few abnormal adults and for a series of 26 normal newborns.     

The effect of anticoagulant, storage temperature and dilution on cord blood hematology parameters over time

The objective of the study was to determine whether selected hematologic parameters measured on umbilical cord blood samples using an automated hematology analyzer (Sysmex XE-2100) were affected by (i) anticoagulant (the specimens were collected in EDTA vs. sodium heparin), (ii) temperature (the specimens were maintained at 4 degrees C vs. room temperature for up to 72 h) and (iii) 1 : 5 dilution vs. undiluted using the manufacturer's diluting solution. Use of heparin, instead of EDTA, had little effect on the hematologic results (n = 8) except for lower platelet and progenitor cell counts. Results were remarkably stable for 72 h at either room temperature or 4 degrees C except for modest red blood cell swelling at 24 h. Specimens of blood diluted at 1 : 5 had an immediate small, but significant change on white cell count (+13.3%), reticulocyte count (-11.2%) and reticulocyte hemoglobin content (-19.6%). Diluted samples did not change further over 4 h at room temperature. With a 1 : 5 dilution, analysis of 40 microl of cord blood stored for 3 days at room temperature may provide useful hematologic information with little phlebotomy loss.     

A method for the determination of the osmotic fragility of erythrocytes

A method for registration of the haemolysis of erythrocytes as a measure for their osmotic resistance is presented. The technical realization is performed by means of a cuvette head-piece for holding a filter, which separates the investigation material from water. A cuvette insertion guarantees the regular construction of a haemoglobin gradient, serving as a measure for haemolysis, which is photometrically measured in the Spekol (VEB Carl Zeiss JENA) and registered by means of the compensation tape writer G 1 B 1 (VEB Carl Zeiss JENA). 0.3-0.4 ml investigation material are used. The expenditure of time amounts to about 20 minutes.     

The osmotic fragility of erythrocytes after prolonged liquid storage and after reinfusion

Although it is recognized that red cells lose membrane during storage, estimation of the osmotic fragility of erythrocytes has not previously proven to be a useful measurement of the storage lesion. Erythrocytes from blood stored in CPD-A2 were found to have a markedly increased osmotic fragility. A major portion of this increase was found to be due to accumulation of lactate, which is only slowly transported from within erythrocytes and which therefore exerts a strong osmotic effect in the usual osmotic fragility test. After an hour's incubation in a large volume of iso-osmotic buffer, the osmotic fragility curve of stored erythrocytes was much more nearly normal. Such cells were found to have a volume 5%--8% greater than that of normal cells, indicating that even after removal of lactate more osmotically active material was present in the stored erythrocytes than in fresh cells. Most of this differences can be accounted for by substitution of chloride ion for 2,3-DPG, since chloride exerts approximately 3.7 times the osmotic effect of 2,3-DPG per unit charge. In addition to the shift in osmotic fragility produced by the increased intracellular osmotically active material, a "fragile tail" of red cells was also present. Stored erythrocytes were labeled with 51Cr and reinfused into the volunteer donors. The osmotic fragility of the reinfused cells was estimated using a technique of sequential osmotic hemolysis that permitted accurate estimation of osmotic fragility of transfused cells using very small amounts of 51Cr. The osmotic fragility of the reinfused cells became less than those of fresh cells after 24 hr and was exactly the same as those of fresh cells after 4 days. The fragile tail disappeared at a rate that approximated the rate of loss of nonviable erythrocytes from the circulation as measured by 51Cr. These findings are consistent with the preferential destruction of a subpopulation of red cells with a diminished surface area.     

Modified osmotic fragility test for the laboratory diagnosis of hereditary spherocytosis

A modified osmotic fragility test, based on measurement of hemolysis in four hypotonic NaCl solutions and logarithmic linearization of osmotic fragility curve is, like the "Pink test," a specific and sensitive test for the laboratory diagnosis of hereditary spherocytosis.     

Cryohemolysis for the detection of hereditary spherocytosis: Correlation studies with osmotic fragility and autohemolysis

Laboratory methods aimed to assess the presence of spheroidal cells such as osmotic fragility, autohemolysis, and glycerol lysis time are very elaborate, time consuming, and often give inconclusive results. We have developed a diagnostic test based on a unique sensitivity of HS cells to hypertonic cryohemolysis and analyzed blood samples of 55 HS patients. The patients were divided into two subgroups, clinically affected probands and their relatives. To get quantitative comparisons with the classic methods, the cryohemolysis results were compared to two parameters of the osmotic fragility test: the salt concentration that causes 50% hemolysis, and the percent lysis at a constant salt concentration. Autohemolysis results were also compared. To evaluate which of these tests has the best analytical power, we calculated the mean results and 2 SDs of each parameter in a control group, and then looked to see which of them was best in identifying the patients. The cryohemolysis test was the single parameter that identified all cases including asymptomatic carriers of the disease. The ability of this test to identify the less severe cases probably reflects the dependency of the cryohemolysis on factors that are more related to the primary membrane molecular defects and less by the surface area to volume ratio. Am. J. Hematol. 58:206–212, 1998. © 1998 Wiley-Liss, Inc.     

联合试验在地中海贫血诊断中的应用

目的 :探讨红细胞平均体积 (MCV)、红细胞脆性及血红蛋白 (Hb)电泳联合试验在地中海贫血 (简称地贫 )中的诊断价值。方法 :用日本SysmexKX 2 1自动血细胞分析仪、中山医科大学红细胞脆性一管定量法、美国Helena公司KEP型全自动电泳分析系统 ,分别对本实验室经分子生物学技术确诊的 14 8例地贫及 81例非地贫样品进行MCV、红细胞脆性及Hb电泳测定 ,并对结果作相关统计学分析。结果 :MCV、红细胞脆性、Hb电泳单项试验对地中海贫血诊断的灵敏度及特异度分别为 91.9%、81.1%、83.8%及 79%、91.4 %、90 .1%。MCV与Hb电泳、红细胞脆性与Hb电泳二项试验平行联合的灵敏度及特异度分别为 98.6 %、96 .6 %及 71.6 %、82 .7% ;系列联合的灵敏度及特异度分别为 77%、6 8.2 %及 97.5 %、98.8%。MCV、红细胞脆性、Hb电泳三项试验平行联合的灵敏度、特异度为 10 0 %、6 5 .4 % ;系列联合的灵敏度及特异度为 6 2 .8%、10 0 %。经u检验 ,平行联合试验的灵敏度与各单项试验灵敏度之间、系列联合试验的特异度与各单项试验特异度之间差异有统计学意义 (P <0 .0 5 )。结论 :在地贫的诊断试验中 ,MCV、红细胞脆性、Hb电泳平行联合试验可提高灵敏度 ,系列联合试验可提高特异度 ,联合试验较单项试验有一定的优越性     

Comparison of acidified glycerol lysis test, Pink test and osmotic fragility test in hereditary spherocytosis: effect of incubation

In this study we compared the results of the acidified glycerol lysis test, the Pink test and the osmotic fragility test in 38 patients with hereditary spherocytosis and in healthy controls. The sensitivity of the acidified glycerol lysis test was 81.6% when performed within 3 h after blood collection. After incubating for 24 h, the sensitivity increased to 100% whereas the specificity remained maximal. Similar incubation did not improve the diagnostic utility of the Pink test. All patients, but none of the controls, showed a positive osmotic fragility test. It is concluded, because of sensitivity and specificity in this study, that the acidified glycerol lysis test after incubation and the osmotic fragility test are superior to the Pink test in detecting spherocytosis.     

Diminished osmotic fragility of human erythrocytes following the membrane insertion of oxygenated sterol compounds

Oxygenated sterol compounds (OSC), when incubated for 1 hr with human erythrocytes in lipoprotein-depleted medium at concentrations of 0.625- 5 X 10(-5) M, are inserted into the cell membrane and remain there despite subsequent washing of the cells. The insertion results in expansion of the surface area of the red cell ghost membrane, an increase in critical hemolytic volume, and as a consequence, in diminished osmotic fragility of the erythrocytes. This effect is seen with echinocyte-forming as well as with non-echinocyte-forming OSC. Erythrocytes treated with OSC do not differ from control cells with respect to their mean cell volume (MCV) in isotonic solution, water content, ion fluxes, and filterability through polycarbonate filters. The shift of the osmotic fragility curve toward lower NaCl concentrations is proportional to the amount of OSC inserted into the red cell membrane. 7 beta-Hydroxycholesterol, 22-ketocholesterol, and 20 alpha-hydroxycholesterol are the most potent inhibitors of osmotic lysis. The effect of OSC on osmotic fragility is diminished if the erythrocytes are incubated in a lipoprotein-containing medium; free cholesterol, however, does not change this effect. Various progesterones also protect red cell from osmotic lysis, but only if the erythrocytes are directly exposed to the compounds present in the hypotonic NaCl solutions used for measurement of their osmotic fragility. Progesterones do not remain in the membrane after the cells have been washed. The OSC are also capable of correcting the osmotic fragility curve of red cells from patients with hereditary spherocytosis. These experiments may suggest an approach to the pharmacologic treatment of hereditary spherocytosis.     

The Impact of Heparin Compounds on Cellular Inflammatory Responses: A Construct for Future Investigation and Pharmaceutical Development

Atherosclerotic disease is recognized as a chronic inflammatory disorder with intermittent and widely variable phases of cellular proliferation and heightened thrombotic activity. The multi-tiered links between inflammation, atherogenesis and thrombogenesis provide a unique opportunity for research and development of pharmaceuticals which target one or more critical pathobiologic steps (Fig. 1).The purpose of the following review on heparin compounds is to comprehensively examine the multi-cellular, pleuripotential effects of a commonly used anticoagulant drug in the context of normal and disease-altered vascular responses and illustrate possible constructs for avenues of subsequent investigation in the field of atherothrombosis.The overview is divided into five integrated parts; antiinflammatory properties of the normal vessel wall, the relationship between glycosaminoglycans and inflammation, heparin-mediated effects on cellular inflammatory responses, association between molecular weight and antiinflammatory capabilities, and oral heparin compounds for achieving prolonged cell-based inhibition.     

Alterations in calcium-handling of erythrocytes in spontaneously hypertensive rats. Calcium-induced changes in the osmotic fragility of erythrocytes in hypertension

To investigate the sensitivity to calcium of erythrocytes in hypertension, changes in the osmotic fragility of erythrocytes following Ca-loading were observed. Washed erythrocytes were obtained from spontaneously hypertensive rats (SHR, Okamoto and Aoki) and age-matched normotensive Wistar Kyoto rats (WKY). Treatment of erythrocytes with Ca-ionophore A23187 and Ca in the medium caused a reduction in the osmotic fragility which correlated with the Ca-concentration. The degree of alteration in the osmotic fragility of erythrocytes was greater in SHR than in WKY. Oral administration of hydralazine to SHR significantly reduced the blood pressure. However, the alterations in the osmotic fragility of erythrocytes secondary to Ca-loading were not different between hydralazine-treated and untreated SHR. In the presence of a Ca-antagonist (verapamil or diltiazem) in the medium, the reduction of the osmotic fragility of erythrocytes caused by Ca-loading was inhibited, and the differences between SHR and WKY were abolished by Ca-antagonists. These results suggest that the greater changes in osmotic fragility of erythrocytes caused by Ca-loading in SHR could be due to a genetic abnormality of Ca-handling by the cell membranes, and that this abnormality might cause an increase in intracellular Ca, which contributes, in part, to the pathogenesis of hypertension.