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.     

Linolenoyl Sorbitol and the Fragility of Hereditary Spherocytes

S ummary . Linolenoyl sorbitol decreased uniformly the osmotic fragility of normal human red cells. When added to red cells of patients with hereditary spherocytosis (HS), the synthetic lipid afforded the most fragile cells the greatest shift in osmotic stability. The greater the osmotic fragility of red cells from different patients, the greater the protective effect afforded by the added lipid. Linolenoyl sorbitol fully reversed the increased fragility of vinblastin-treated red cells. The results indicate a causal relationship between the lipid composition and the increased fragility of red cells in hereditary spherocytosis.     

Erythrocyte cellular and membrane deformability in hereditary spherocytosis

In order to determine whether the relative rigidity of the hereditary spherocytosis (HS) red cell is due to membrane rididity or merely to an altered surface/volume ratio, we investigated the deformability of resealed red cell membranes from patients with HS. Whereas the osmotic fragility of intact red cells of HS patients showed the expected increase, the osmotic fragility of resealed HS membranes was normal, thus indicating that their surface/volume ratio was normal. Measurements with an ektacytometer showed that deformability of intact HS cells was markedly diminished, whereas deformability of resealed HS membranes was normal. These findings indicate that the HS red cell membrane is not intrinsically abnormally rigid, as has been suggested, but that the lack of deformability of the erythrocyte is primarily a function of the altered surface/volume ratio.     

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%).     

Studies on spontaneous in vitro autohemolysis in hemolytic disorders

Measurements of spontaneous lysis (autohemolysis) of red cells in sterile defibrinated blood after 48 or more hours of incubation at body temperature werefound useful in the investigation of certain hemolytic states. Abnormally rapidautohemolysis was demonstrated most consistently in hereditary spherocytosis,but was also found in other types of spherocytosis and in several examples ofnon-spherocytic hemolytic anemia. Autohemolysis above the normal range canbe regarded as strong presumptive evidence of a hemolytic disorder, but a normalrate of autohemolysis by no means excludes the possibility of a hemolytic process in any given case.

Abnormalities of hereditary spherocytes causing increased autohemolysis wereshown to be correlated with those responsible for spherocytosis as reflected inosmotic fragility tests. There appeared to be closer correlation with abnormalities causing increased osmotic fragility of the cells after 24 hours incubation.The autohemolysis test and the osmotic fragility test on incubated red cells werefound to be equally sensitive in their capacity to detect spherocytosis of thehereditary type.

Addition of adenosine, guanosine or inosine caused moderate to marked reduction in autohemolysis of nearly all types of red cells tested. Lysis of incubatedspherocytes from active cases of autoimmune hemolytic disease was more markedly inhibited by a small amount of adenosine than was the lysis of hereditaryspherocytes.

Glucose regularly caused marked inhibition of autohemolysis of hereditaryspherocytes and of red cells from patients with acute leukemia. The effect ofglucose on HS red cells might have been due in part to a lowering of pH by formation of lactic acid since acidification of the blood with lactic, citric or hydrochloric acid also caused substantial reduction in autohemolysis.

Glucose caused slight to marked increase in lysis of red cells in certain othercases of spherocytosis, notably in autoimmune hemolytic disease and in myeloidmetaplasia. Addition of lactic acid to the blood from several of these patientshad similar effect.

In 2 atypical cases of chronic spherocytosis, with hematologically normalrelatives, autohemolysis was accelerated by addition of glucose to the bloodsamples. Adenosine had little effect until after splenectomy when both glucoseand adenosine inhibited hemolysis, much the same as in blood from typicalcases of hereditary spherocytosis.

It seems likely that when the abnormalities of carbohydrate metabolism ofthe red cells in certain hemolytic disorders are better understood, measurementsof autohemolysis may be modified so as to enhance their usefulness in detectingand differentiating the various hemolytic states. It also seems likely that furtherstudies on in vitro autohemolysis will help to elucidate some hemolytic mechanisms operating in vivo.

Submitted on March 14, 1956 Accepted on April 17, 1956     

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.     

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.     

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.     

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.     

Production of Spherocytosis and Increased Osmotic Fragility in Normal Erythrocytes by Sodium Stress

Red cells from normal human subjects were exposed to hypertonic sodium chloride solutions for varying periods of time. The cell changes commonly observed in hereditary spherocytosis were induced, including microspherocyte formation with corresponding increase in osmotic fragility. Solutions of magnesium sulphate of equal osmolarity did not produce these changes. These observations lend support to the theory that excessive sodium permeability is concerned in the pathogenesis of hereditary spherocytosis. Increased influx of sodium and water into erythrocytes leads to spherocytosis, but only increased efflux of sodium leads to irreversible microspherocytosis.     

Atypical Hereditary Spherocytosis: Biochemical Studies and Sites of Erythrocyte Destruction

A family with hereditary spherocytosis has been investigated and found to have the following unusual features: atypical pre- and post-incubation red cell osmotic fragility and absence of splenic sequestration of red cells. Ouabain, an inhibitor of active cation transport, was found not to inhibit the corrective effect of glucose on the rate of autohaemolysis of whole blood, a finding present in some cases of known hereditary spherocytosis, but not in others. The red cells of the propositi showed evidence of increased A.T.P.-ase activity as well as increased utilization of A.T.P. for purposes other than for active cation transport, and are metabolically similar to hereditary spherocytic cells in this respect. The reason for the absence of splenic sequestration of red cells was not established.     

Failure of Ouabain to Influence the Osmotic Fragility in Hereditary Spherocytosis

No effect of 3 μmol ouabain on the osmotic fragility of red blood cells in subjects suspected of being carriers of hereditary spherocytosis, as well as in patients with overt disease could be demonstrated. These results are in disagreement with a recent report. Some possible explanations for these discrepant results are discussed. It is concluded that ouabain probably adds little to the diagnostic capability of the osmotic fragility test.     

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.     

A new test for the laboratory diagnosis of spherocytosis

A new test for the laboratory diagnosis of spherocytosis, conventionally called 'Pink test', is presented. This test, semi-quantitatively or quantitatively, determines the hemolysis of small blood samples in a solution containing glycerol (135 mmol/l), NaCl (25 mmol/l), NaN3 (1.5 mmol/l), buffered to pH 6.66 with Bis-Tris (70 mmol/l) and HCl. 'Pink test', as well as 'acidified' glycerol lysis test, were positive in 100% of 42 patients suffering from hereditary spherocytosis, and optimally discriminated them from healthy subjects, showing a diagnostic sensitivity greater than 'standard' glycerol lysis test and osmotic fragility in hypotonic saline solutions of fresh or incubated blood. 'Pink test' was also positive in some cases of renal failure, immunohemolytic anemia, chronic hemoproliferative disorders, normal pregnant women, and negative in other microcytic anemias (beta-thalassemia, iron deficiency anemia). The results do not critically depend on pH of the solution (differently from those obtained with 'acidified' glycerol lysis test), and for this reason they show a good reproducibility.     

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 polycarbonate 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.     

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.     

Hypertonic cryohemolysis: a diagnostic test for hereditary spherocytosis

Red blood cells of subjects with hereditary spherocytosis are specifically susceptible to temperature changes while suspended in hypertonic solutions. Based on this property, a new diagnostic test for hereditary spherocytosis is presented. The suggested method is 100% sensitive in the diagnosis of all patients, including asymptomatic obligate carriers with hereditary spherocytosis, and is very specific in different control groups. Unlike other methods designed for the diagnosis of hereditary spherocytosis, this test does not depend on the cells' surface- area-to-volume ratio. Normal red blood cells that were induced to become spherocytes by different means (i.e., vinblastine, lysophosphatidyl choline, and heat) and showed increased osmotic fragility did not become susceptible to the hypertonic cryohemolysis conditions.     

Haematological Changes in Heat-Acclimated Golden Hamsters

S ummary . Haematological changes were investigated in heat-acclimated hamsters. Haemoglobin, packed-cell volume, and red-cell counts were not affected by heat acclimation. Glucose uptake and osmotic fragility in the red blood cells of these animals were increased. The red cell ATP level was lower in heat-acclimated than in control hamsters. No change was observed in the density distribution of the red cell population in heat-acclimated hamsters. The resemblance of these cells to the red cells of hereditary spherocytosis and pyruvate-kinase deficiency is discussed.     

Hereditary spherocytosis. I. Clinical,hematologic and genetic features in 28 cases,with particular reference to the osmotic and mechanical fragility of incubated erythrocytes

Clinical, hematologic and genetic data on 28 cases of hereditary spherocytosisare presented for the purpose of characterizing this disorder as completely aspossible. On the basis of this experience it is recommended that the followingtypical laboratory findings be sought in establishing a diagnosis in suspectedcases: (1) Presence of spherocytes or abnormally thick red cells in peripheralblood; (2) greater than normal osmotic fragility of the red cells; in cases in whichthe fragility of fresh cells is not significantly increased, determinations should bemade after sterile incubation of the blood at body temperature for 24 hours;(3) greater than normal mechanical fragility of freshly drawn red cells; (4) negative antiglobulin (Coombs) test; (5) greater than normal lysis of the red cellsduring sterile incubation at body temperature for 48 hours; and (6) presence ofsimilar abnormalities in relatives.

Abnormality of the erythrocyte persisted in all of the 11 patients in this seriesfollowed one or more years after splenectomy. An unusual case of chronic hemolytic anemia is described but not included in the numbered series because (1) bothparents were hematologically normal and (2) spherocytosis and abnormally greatosmotic and mechanical fragility and autohemolysis could not be demonstratedafter the fifth postoperative month. Classification of this case is deferred pendingfurther experience.

Demonstration in a parent, sibling or offspring of red cells showing the afore-mentioned abnormalities is necessary for an unequivocal diagnosis, but this requirement cannot always be met because relatives may not be available for examination. Moreover, when parents and/or several siblings are examined withoutpositive findings, low gene expressivity, gene mutation and illegitimacy may beconsidered as explanations. Evidence is cited to suggest the possibility of a lowdegree of penetrance or expression in some cases and to illustrate the need forstill more sensitive laboratory tests that might aid in diagnosis of the mildestforms of this disease. The lower incidence of spherocytosis in siblings of propositithan in offspring of propositi is cited as evidence bearing on the theory of genemutation in some propositi.

A simplified "qualitative" test of osmotic fragility of incubated red cells isdescribed.

Submitted on April 16, 1951 Accepted on May 10, 1951     

Hereditary Spherocytosis: Observations on Hemolytic Mechanisms and Iron Metabolism

1. Two healthy patients with hereditary spherocytosis were phlebotomizeduntil they developed iron deficiency and the erythrocytes became hypochromic.The hereditary spherocytes were no longer spheroidal: they became thin, andthe fragility tests improved. However, the life span of the cells in the circulation was not improved. Later, splenectomy corrected the hemolytic disease.

2. In both patients, prior to the experiment, the hemolytic disease wascompensated. There was no anemia despite the rapid turnover of red cells.In one of the patients, whose average red cell life span was only five days, theoutput of hemoglobin must have been exceedingly high. It was computed tobe 135 Gm. per day, or 20 times the normal rate.

3. Some aspects of iron metabolism in hereditary spherocytosis are discussed.

4. The shape of the red cell in HS does not appear to be responsible for itspremature destruction by the spleen. Iron deficiency corrects the spherocytosis,but it does not correct the hemolytic disease. Splenectomy corrects the hemolytic disease, but it does not correct the spherocytosis.