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%).     

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.     

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.     

Diagnostic utility of the pre-incubated acidified glycerol lysis test in haemolytic and non-haemolytic anaemias.

The usefulness of the pre-incubated acidified glycerol lysis test (AGLT), a laboratory test for spherocytosis, has been investigated in a selected hospital population of 348 patients with haemolytic and non-haemolytic anaemia. The AGLT was positive in 58 out of 59 patients with hereditary spherocytosis. In all 32 patients with other types of hereditary haemolytic anaemia the AGLT was normal or equivocal, but clearly different from spherocytosis. Adults with a positive AGLT, but without hereditary spherocytosis, had auto-immune haemolytic anaemia, myelodysplastic syndrome or were pregnant women. In newborn infants the AGLT was positive, in the first week of life, in those babies having hereditary spherocytosis or immune haemolysis due to blood group incompatibility; no positive AGLT results were seen if no haematological explanation for neonatal hyperbilirubinaemia could be found. At the optimal cut-off point the sensitivity of the AGLT for hereditary spherocytosis was 98.3% and the specificity 91.1%, under the most unfavourable conditions. The AGLT is a very useful and simple test for the diagnosis of hereditary spherocytosis.     

Osmotic gradient ektacytometry: A valuable screening test for hereditary spherocytosis and other red blood cell membrane disorders

Introduction

New generation osmotic gradient ektacytometry has become a powerful procedure for measuring red blood cell deformability and therefore for the diagnosis of red blood cell membrane disorders. In this study, we aim to provide further support to the usefulness of osmotic gradient ektacytometry for the differential diagnosis of hereditary spherocytosis by measuring the optimal cutoff values of the parameters provided by this technique.

Methods

A total of 65 cases of hereditary spherocytosis, 7 hereditary elliptocytosis, 3 hereditary xerocytosis, and 171 normal controls were analyzed with osmotic gradient ektacytometry in addition to the routine red blood cell laboratory techniques. The most robust osmoscan parameters for hereditary spherocytosis diagnosis were determined using receiver operating characteristic curve analysis.

Results

The best diagnostic criteria for hereditary spherocytosis were the combination of decreased minimal elongation index up to 3% and increased minimal osmolality point up to 5.2% when compared to the mean of controls. Using this established criterion, osmotic gradient ektacytometry reported a sensitivity of 93.85% and a specificity of 98.38% for the diagnosis of hereditary spherocytosis.

Conclusion

Osmotic gradient ektacytometry is an effective diagnostic test for hereditary spherocytosis and enables its differential diagnosis with other red blood cell membrane diseases based on specific pathology profiles.     

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.     

The effect of splenectomy in hereditary spherocytosis by dual angle laser light cytometry

Summary We have applied dual angle laser scattering cytometry (DALC), which provides objective assessment of spherocytosis, to study the changes of hereditary spherocytosis (HS) red cell populations after splenectomy. Eighty unsplenectomized HS patients (32 mild, 37 moderate and 11 severe cases), 26 splenectomized HS patients (SHS, formerly 21 moderate and five severe cases) and 140 controls were studied. SHS were similar to unsplenectomized mild HS cases, with homogeneous red cell volume distribution and spherocytosis. Spherocytosis in SHS was significantly higher when compared with controls (P < 0.001) but less when compared with unsplenectomized HS patients (P < 0.01).     

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     

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.     

Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics

Background

The laboratory diagnosis of hereditary spherocytosis commonly relies on NaCl-based or glycerol-based red cell osmotic fragility tests; more recently, an assay directly targeting the hereditary spherocytosis molecular defect (eosin-5′-maleimide-binding test) has been proposed. None of the available tests identifies all cases of hereditary spherocytosis.

Design and Methods

We compared the performances of the eosin-5′-maleimide-binding test, NaCl-osmotic fragility studies on fresh and incubated blood, the glycerol lysis test, the acidified glycerol lysis test, and the Pink test on a series of 150 patients with hereditary spherocytosis grouped according to clinical phenotype and the defective protein, with the final aim of finding the combination of tests associated with the highest diagnostic power, even in the mildest cases of hereditary spherocytosis.

Results

The eosin-5′-maleimide-binding test had a sensitivity of 93% and a specificity of 98% for detecting hereditary spherocytosis: the sensitivity was independent of the type and amount of molecular defect and of the clinical phenotype. The acidified glycerol lysis test and Pink test showed comparable sensitivity (95% and 91%). The sensitivity of NaCl osmotic fragility tests, commonly considered the gold standard for the diagnosis of hereditary spherocytosis, was 68% on fresh blood and 81% on incubated blood, and further decreased in compensated cases (53% and 64%, respectively). The combination of the eosin-5′-maleimide-binding test and acidified glycerol lysis test enabled all patients with hereditary spherocytosis to be identified. The eosin-5′-maleimide-binding test showed the greatest disease specificity.

Conclusions

Each type of test fails to diagnose some cases of hereditary spherocytosis. The association of an eosin-5′-maleimide-binding test and an acidified glycerol lysis test enabled identification of all patients with hereditary spherocytosis in this series and, therefore, represents a currently effective diagnostic strategy for hereditary spherocytosis including mild/compensated cases.     

Membrane cation and anion transport activities in erythrocytes of hereditary spherocytosis: Effects of different membrane protein defects

Hereditary spherocytosis (HS) is due to different membrane protein defects (i.e., deficiency of spectrin and ankyrin, band 3, or band 4.2). In order to gain new insight into the relationships between band 3 function and proteins associated with the cytoskeleton, we studied erythrocyte anion transport activity in HS characterized by different membrane protein defects. Anion transport activity was increased in HS due to partial band 4.2 deficiency or to band 4.2 absence, while in HS associated with deficiency of spectrin + ankyrin or band 3, the anion transport results were normal or decreased, respectively. Moreover, since HS erythrocytes are characterized by an increased Na and a decreased K, we studied the principal membrane cation transport pathways. Activity of the Na/K pump was increased in all HS studied, while no changes in Na/K/2Cl cotransport and Na/Li exchange were evident between control and HS as well as between forms of HS associated with different membrane protein defects. K/Cl cotransport activity was decreased in all HS studied compared to normal red cells. In all HS, passive membrane permeability to Na and K was increased compared to normal erythrocytes. The increased Na and the low K content can be attributed to the abnormal membrane permeability to cations, which is not related to a specific membrane protein defect. Am. J. Hematol. 55:121–128, 1997. © 1997 Wiley-Liss, Inc.     

Frequent de novo mutations of the ANK1 gene mimic a recessive mode of transmission in hereditary spherocytosis: three new ANK1 variants: ankyrins Bari, Napoli II and Anzio

A subset of spherocytosis cases associated with mutations of the ANK1 gene present an apparently recessive inheritance pattern on a clinical and haematological basis. We identified three novel out-of-frame deletions in the ANK1 gene: allele Bari (1361delG), Napoli II (2883delC) and Anzio (3032delCA) in three Italian patients, two of whom have been splenectomized. Analysis of the cDNA showed small or trace amounts of ankyrin mRNAs in Bari, Napoli II and Anzio. The parents were normal clinically and haematologically and did not carry the mutations exhibited by their children. We confirmed the de novo character of the HS mutations based on paternity testing. Recessive HS associated with the ANK1 gene is probably rarer than initially thought, and spherocytosis may often be due to de novo mutations.     

Elevated levels of redox regulators, membrane-bound globin chains, and cytoskeletal protein fragments in hereditary spherocytosis erythrocyte proteome

Objective: Hereditary spherocytosis (HS), a common inherited hemolytic anemia characterized by decreased deformability, reduced surface to volume ratio, and increased osmotic fragility of the spheroidal erythrocytes, is associated with several mutations of α‐ and β‐spectrin, ankyrin, band 3, band 4.2. HS manifests itself with high degrees of clinical heterogeneity and the molecular events leading to premature hemolysis of the spherocytes are unclear. We have employed proteomic techniques to identify differentially regulated proteins in the membrane and hemoglobin‐depleted cytosol of HS erythrocytes. Methods: We have employed 2‐D gel electrophoresis and tandem matrix assisted laser desorption ionization‐time of flight/time of flight mass spectrometry to investigate the differential proteome profiling of membrane and hemoglobin‐depleted cytosol of erythrocytes isolated from the peripheral blood samples of HS patients and normal volunteers. Results: Our study showed that redox regulators are up‐regulated; while a co‐chaperone and a nucleotide kinase are down‐regulated in HS erythrocyte cytosol. We observed elevated levels of membrane‐associated globin chains and low‐molecular weight fragments of several major cytoskeletal proteins. Conclusion: The observed changes in the erythrocyte proteomes indicate altered redox regulation, nucleotide metabolism, protein aggregation and/or degradation, cytoskeletal disorganization, and severe oxidative stress in HS. Taken together, this study could enlighten upon disease progression and pathophysiology of HS.