Does Preincubation of the Red Blood Cells Contribute to the Capability of the Osmotic Fragility Test to Detect very Mild Forms of Hereditary Spherocytosis?

During incubation for 24 h at 37°C, erythrocytes from patients with hereditary spherocytosis (HS) undergo a greater increase in osmotic fragility than do normal cells, and this procedure has been recommended for differentiating more clearly between patients with very mild HS and normal subjects. The greater effect of preincubation on erythrocytes from patients with HS was confirmed, but, except in cases demonstrating a markedly increased osmotic fragility before incubation, this effect was outweighed by a simultaneous loss of test precision. It therefore seems that preincubation does not significantly contribute to the capability of the osmotic fragility test to detect very mild forms of HS.     

Effect of Ouabain on Osmotic Resistance and Monovalent Cation Transport of Red Cells in Hereditary Spherocytosis

The effect of ouabain on the osmotic resistance of red cells from 17 splenectomized patients with congenital or hereditary spherocytosis (HS), from 5 of their relatives suspected of having a subclinical form of the disease and from unsplenectomized and splenectomized normal controls was studied. In red cells from the HS patients and the splenectomized controls a small ouabain-induced decrease in osmotic resistance was seen, whereas in the unsplenectomized controls no such change was observed. The osmotic resistance of red cells from the 5 relatives, on the other hand, was significantly decreased by ouabain. The usefulness of ouabain in unmasking HS in its subclinical form was further tested by studying monovalent cation influx into red cells. The active influx of ⁸⁶Rb+ into red cells from HS sufferers was significantly increased. A similar degree of ouabain-induced inhibition of ⁸⁶Rb+ transport was seen in all groups studied. The passive influx of ²²Na+, which was also increased in HS red cells, was not affected by ouabain in any of the four groups.     

Haemolysis in Athletes due to Hereditary Spherocytosis

Three athletes, one cross-country skier and two middle distance runners, experiencing poor response to intensified training and decline in performance during prolonged periods of frequent competitions, associated with a tendency to develop anaemia, were found to have spherocytosis and increased osmotic fragility of the red blood cells. All had family members with the same blood abnormality, but without symptoms. The observations suggest that the frequency of hereditary spherocytosis is higher than indicated by overtly affected cases and that mild cases are easily overlooked. Hereditary spherocytosis should be looked for in athletes with a tendency to decline in blood haemoglobin concentration during periods of intensive training and competition.     

Lineation of the Osmotic Fragility Curve of Erythrocytes

Lineation of the osmotic fragility curve by the method of Detraglia et al (1974) may be performed in most samples from a hospital population. Using the lineation procedure, the osmotic fragility may be tested by only 2 solutions of known osmolarity without any great loss of precision or accuracy. The osmotic fragility curve may be described by 2 values: C₅₀ = the concentration at which 50% of the erythrocytes are haemolyzed and C₅₀ - C₈₀ = the decrease in concentration raising the fraction of haemolysis from 0.50 to 0.80.     

Ageing of Blood in Hereditary Spherocytosis

Blood from 5 patients suffering from hereditary spherocytosis (HS) was stored in acid-citrate-dextrose anticoagulant at 4°C for up to 6 weeks. The erythrocyte morphology and susceptibility to lysis by phospholipase C (Bacillus cereus) were examined at 2-weekly intervals and compared with erythrocytes from 6 different donations of stored normal blood. The hereditary spherocytes went through essentially the same series of morphological changes as did normal erythrocytes. Fresh hereditary spherocytes were very resistant to lysis by phospholipase C, but, like normal erythrocytes, became progressively more susceptible to lysis upon storage. In terms of erythrocyte morphological changes and development of susceptibility to lysis by the enzyme, blood from HS patients aged less rapidly than did normal blood. Splenectomy appeared to have no marked effect on the storage properties of blood from these patients as indicated by erythrocyte morphology and susceptibility to lysis by phospholipase C.     

Osmotic Erythrocyte Fragility and ABO Blood Groups

In a study of 1008 blood donors a reduced frequency of blood group A and an increased frequency of blood group O were observed in those with increased osmotic fragility of their red cells.     

The Normal Range of Osmotic Fragility of Red Blood Cells

With the technique of Parpart et al (1947), the normal range for osmotic fragility of red blood cells has been estimated to include 5%–45% haemolysis at a salt concentration corresponding to 4.5 g NaCl/1 (Dacie 1954). This estimate may be questioned, however. Thus, nearly 20% of the data obtained from 50 presumably healthy subjects fell outside these limits. Furthermore, the distribution pattern was very asymmetric with erroneously small standard deviation. On the other hand, if the technical conditions (salt concentration, buffer) were properly adjusted, nearly all the observations were located within the straight part of the s-shaped haemolysis curve and scattered symmetrically around the mean. Under these conditions, the normal range (mean ± 2 SD) included 22%–88% haemolysis. Moreover, this wider range included about 95% of the observations, even adapted to the original experimental situation. The present estimates should therefore replace earlier statements in the literature.     

High Prevalence of Increased Osmotic Fragility of Red Blood Cells among Norwegian Blood Donors

Increased osmotic fragility of red blood cells was found in 9 out of 1008 Norwegian blood donors. In addition, increased osmotic fragility was found in 3 out of 23 first grade relatives and in 1 out of 4 spouses of individuals with the same condition. Finally, there was a positive correlation between increased osmotic fragility and morphological signs of spherocytosis (P < 0.05). No definite conclusions with respect to underlying mechanism(s) for this high prevalence of non-symptomatic increased osmotic fragility can be offered, but very mild hereditary spherocytosis, environmental factors and even a normal variant, never associated with haemolysis, may have contributed. Furthermore, until more specific and sensitive laboratory techniques have been introduced, a proper distinction between these 3 conditions cannot be made.     

The Osmotic Fragility of Red Blood Cells: A Re-Evaluation of Technical Conditions

The osmotic fragility of red blood cells is influenced by even modest environmental changes. Consequently, the technical procedure must be strictly standardized. This implies that temperature equilibrium of the buffered salt solutions should be reached prior to the addition of blood. Furthermore, since erroneous statements concerning the composition of phosphate buffers regularly used to secure correct pH of the salt solutions have repeatedly appeared in the literature, pH control of such solutions prior to use becomes essential.     

Effect of Pentoxifylline on Red Cell Flexibility and Cation Transport in Healthy Subjects and Patients with Hereditary Spherocytosis

The effect of pentoxifylline (3,7-dimethyl-1-(5-oxo-hexyl)-xanthine) on the flexibility of red cells was studied using a filtration method in which the red cells are forced, at a constant flow-rate, through a porous pdycarbonate membrane. The filtration pressure reflects red cell rigidity and the amount of Hb released from the disrupted cells (‘free Hb’) red cell fragility. The advantage of this method is that it allows the two important determinants of red cell flexibility, rigidity and fragility, to be studied simultaneously. Pentoxifylline significantly improved normal red cell flexibility both in vivo and in vitro as judged by this method. The impaired flexibility of red cells from patients with congenital or hereditary spherocytosis (HS) was aggravated by pentoxifylline in vitro. A similar effect of pentoxifylline was also observed on red cells from relatives of HS patients. The effect of pentoxifylline on Ca²⁺ transport and Ca²⁺Mg²⁺-ATPase activity in red cells from normal and HS patients was investigated. Under in vitro conditions pentoxifylline did not affect the Ca²⁺Mg²⁺-ATPase activity of or Ca²⁺ efflux from normal and HS red cells. Neither the influx of monovalent cations (Na+, Rb+) or the osmotic resistance of normal or HS red cells was affected by pentoxifylline.     

Hereditary Spherocytosis in Greece: Collective Data on a Large Number of Patients

This paper summarizes data from a haematological, biochemical, and clinical study carried out in 73 patients of Greek origin (38 non-splenectomized children and 35 adults; 17 splenectomized) with Hereditary Spherocytosis (HS). Mean haemoglobin levels in the non-splenectomized patients were significantly lower (122 ± 15 g/L) than those of the splenectomized group (144 ± 15 g/L). In all patients with HS (non-splenectomized and splenectomized adults, and children) the MCHC values (355 ± 22, 358 ± 16 and 356 ± 16 respectively) were significantly increased compared to a control group, while the percentage of microcytic and hyperchromic red cell subpopulations was significantly increased in the former group of adults. SDS-PAGE demonstrated that 29 patients (39.7%) had isolated spectrin deficiency, 22 patients (30.1%) had combined spectrin and ankyrin deficiency, 17 patients (23.3%) had band 3 deficiency and 1 patient had protein 4.2 deficiency. No quantitative biochemical defects were detected in 4 patients (5.5%). The biochemical findings did not correlate with the haematological and clinical phenotype of the disease.     

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.     

A Novel SPTA1 Mutation in a Patient with Hereditary Spherocytosis without a Family History and Coexisting Gilbert's Syndrome

Most patients with hereditary spherocytosis (HS) have a family history of disease, while those without such a history are difficult to diagnose. We herein report a case of HS with no family history harboring a novel heterozygous mutation of SPTA1, c.2161G>A (p.E721K), and a homozygous polymorphism of UGT1A1*6. In silico analyses suggested that the mutation might contribute to the pathogenesis of HS. The coexistence of HS and Gilbert''s syndrome increases the risk of gallstones. Therefore, splenectomy, alone or in combination with cholecystectomy, is recommended. The determination of genetic diathesis provides useful information for the management of hemolytic anemia.     

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.     

Splenic Infarction after Epstein-Barr Virus Infection in a Patient with Hereditary Spherocytosis

We describe the first patient with hereditary spherocytosis (HS) known to have developed splenic infarction following infectious mononucleosis (IM). An 18-year-old Japanese man was referred to our hospital in November 2004 because of continuous fever and icterus. He had undergone cholecystectomy at the age of 14 years. On patient admission in November 2004, a physical examination showed marked hepatosplenomegaly, icterus, and jaundice. He had a white blood cell count of 14.9 x 10(9)/L with 9.5% atypical lymphocytes, a red blood cell count of 2.93 x 10(12)/L, and a hemoglobin concentration of 7.8 g/dL. Microspherocytes were observed in the patient's peripheral blood smear, and immunoglobulin M antibody to Epstein-Barr virus (EBV) viral capsid antigen was detected. The patient's diagnosis was HS with IM. On day 4 of admission, the patient complained of severe abdominal pain. Abdominal computed tomography scanning revealed findings of splenic infarction. Two months after the occurrence of splenic infarction, a splenectomy was performed. A pathohistologic examination of the resected spleen revealed no evidence of thrombosis or arterial occlusion. We assume that the cause of splenic infarction was insufficient blood flow to oxygenate the entire spleen during the acute enlargement of the spleen.     

Increased Osmotic Fragility of Erythrocytes in Essential Hypertension

The correlation between hypertension and the osmotic fragility of erythrocytes was examined. High osmotic fragility of erythrocytes was observed in patients with essential hypertension and normotensive subjects with family history of hypertension, compared with normotensive controls without family history of hypertension. In patients with secondary hypertension, the osmotic fragility of erythrocytes was not significantly different from that of normotensive controls without family history of hypertension. The membrane fragility had no correlation with the level of blood pressure or dietary salt intake. Thus, the osmotic fragility of erythrocytes might reflect functional or structural abnormalities of cell membranes, and could be one of the genetic markers of the hypertensive predisposition.     

Extramedullary Hematopoiesis in Hereditary Spherocytosis Deficient in Ankyrin: A Case Report

Hereditary spherocytosis (HS) is a common inherited hemolytic anemia due to red cell membrane defects. Extramedullary hematopoiesis is a compensatory response to insufficient bone marrow blood cell production. The preferred sites of extramedullary hematopoietic involvement are the spleen, liver, and lymph nodes, but in HS the posterior paravertebral mediastinum is also commonly involved. A nonsplenectomized 74-year-old man with mild HS, with primary deficiency in ankyrin, was found by magnetic resonance imaging to have thoracic paravertebral hematopoietic masses. The patient showed high serum levels of erythropoietin, which may have played a role in the development of extramedullary hematopoietic masses through a continuous hematopoietic stimulus to erythroid cells in the propositus. The long-standing history of respiratory infections and of hypoxia in the propositus may have been an additional etiological factor.