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ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 3  |  Page : 109-113

The application of eosin maleimide-binding test in the diagnosis of hereditary spherocytosis among undiagnosed cases of chronic hemolytic anemia in children


1 Department of Clinical Pathology, Medical Research Institute, University of Alexandria, Alexandria, Egypt
2 Department of Pediatrics, Faculty of Medicine, Medical Research Institute, University of Alexandria, Alexandria, Egypt
3 Department of Hematology, Medical Research Institute, University of Alexandria, Alexandria, Egypt

Date of Submission15-Jul-2014
Date of Acceptance12-Jul-2014
Date of Web Publication31-Dec-2014

Correspondence Address:
Sarah M Nawar
Alexandria Medical Research Institute, 9B Mahmoud Hamdy Khatab st, 5th floor, appartement 502, El Shalalat, Alexandria 21111
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.148229

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  Abstract 

Background Conventional diagnosis of hereditary red blood cell (RBC) membrane disorders, in particular hereditary spherocytosis (HS), is labor intensive, time consuming and requires at least 2 ml of blood, which might be impractical in the neonatal period.
Participants and methods We evaluated the use of eosin-5-maleimide (EMA) as a rapid screening test for patients with HS. RBCs from 74 healthy controls and 66 anemic children (35 HS and 31 other hemolytic anemias; 10 cases diagnosed as thalassemia, eight cases of autoimmune hemolytic anemia, one case of ovalocytosis and 12 cases of undiagnosed hemolytic anemia) were stained with EMA and analyzed for their mean fluorescence intensity using flow cytometry.
Results RBCs from patients with HS showed a greater degree of reduction in mean fluorescence intensity of EMA compared with those from normal controls and patients with other hemolytic diseases. These findings showed that the fluorescence flow cytometric-based method is a simple, sensitive and reliable diagnostic test for RBC membrane disorders using a small volume of blood, and results could be obtained within 2 h. Such a method could serve as a first-line screening for the diagnosis of HS in routine hematology.

Keywords: eosin-5-maleimide, flow cytometry, hereditary spherocytosis


How to cite this article:
El Gendy WM, Hassab HM, Ghanem AM, Lewis IM, Nawar SM. The application of eosin maleimide-binding test in the diagnosis of hereditary spherocytosis among undiagnosed cases of chronic hemolytic anemia in children. Egypt J Haematol 2014;39:109-13

How to cite this URL:
El Gendy WM, Hassab HM, Ghanem AM, Lewis IM, Nawar SM. The application of eosin maleimide-binding test in the diagnosis of hereditary spherocytosis among undiagnosed cases of chronic hemolytic anemia in children. Egypt J Haematol [serial online] 2014 [cited 2017 Oct 17];39:109-13. Available from: http://www.ehj.eg.net/text.asp?2014/39/3/109/148229


  Introduction Top


Hereditary spherocytosis (HS) (also known as the Minkowski-Chauffard syndrome) is a genetically transmitted disorder. It exhibits incomplete penetrance in its expression. It is characterized by the production of red blood cells that are sphere shaped rather than biconcave disk shaped, and therefore more prone to hemolysis [1] .

HS is the most common hemolytic anemia in the western population. It has also been reported in Japanese and African populations. However, the actual incidence of the disease may be significantly higher than reported, as asymptomatic nonpresenting cases may be four to five times more common. The incidence of the disease has not been surveyed widely in some areas, nor have its exact molecular and inheritance patterns been investigated widely [2] .

HS results from the deficiency or dysfunction of one of the red blood cell membrane proteins such as a spectrin, spectrin, ankyrin, an anion channel protein (band-3 protein), protein 4.1 and protein 4.2 [3] .

In HS patients, the family history is often positive for anemia, gallstones or splenomegaly. The majority of the patients (60-75%) have incompletely compensated hemolysis and mild-to-moderate anemia. It is often difficult for parents to recognize the general symptoms of anemia (fatigue, mild pallor or nonspecific findings such as 'crabbiness'), and usually the true extent of reduced physical ability and school deficits become apparent only when positive behavioral changes follow splenectomy. Anemia is the most frequent complaint (50%), followed by splenomegaly and jaundice (all 10-15%) [3] .

The typical laboratory hallmark of HS is the presence of spherocytes that are readily identified by their characteristic shape on the peripheral blood film. They lack central pallor, their mean cell diameter is decreased and they appear to be more intensely hemoglobinated [4] .

HS is characterized by minimal or no anemia, reticulocytosis, an increased mean corpuscular hemoglobin concentration (MCHC), spherocytes on the peripheral blood smear, hyperbilirubinemia and abnormal results on the osmotic fragility (OF) test.

The diagnosis of HS and other membrane disorders is conventionally carried out by a series of investigations, which include peripheral smear examination, reticulocyte count, red cell indices and tests that exploit the surface area-to-volume ratio, which is typically reduced in spherical-shaped erythrocytes, in particular the red cell OF tests at various sodium chloride concentrations on fresh and incubated blood, and assays that measure the extent or the rate of lysis of red cells suspended in buffered glycerol solutions, that is glycerol lysis, acidified glycerol lysis and Pink test [5] .

Although OF has long been considered as the gold standard for diagnosing HS, it requires a large volume of blood and an incubation period of 24 h. It is also not specific for HS. Increased OF may also be seen in immune-mediated hemolysis and a normal OF can be seen in 10-20% of cases of HS [6] .

Among the diagnostic methods considered, a flow cytometry-based analysis by measuring the fluorescence intensity of red cells labeled with eosin-5-maleimide (EMA) dye, which reacts covalently with lysine-430 on the first extracellular loop of band-3 protein, has been developed [6] .

The N-terminal cytoplasmic domain of band-3 interacts with ankyrin and protein 4.2, which interact with the spectrin-based cytoskeleton and stabilize the membrane lipid bilayer. Absent or decreased expression of red blood cell membrane proteins found in HS causes a disruption of the cytoskeleton network and reduces the normal expression of band-3 protein at the erythrocyte membrane. This results in a reduced binding of EMA to band-3 protein and its fluorescence emission [7] .

This study reports the application of this technique in diagnosis of HS among other hemolytic diseases that are prevalent in Egypt including, autoimmune hemolytic anemia (AIHA) which is commonly presented with microspherocytes in blood smear.


  Participants and methods Top


Participants

Thirty-five HS patients were included in this study. HS diagnosis was based on family history, clinical features (anemia, jaundice and splenomegaly), the presence of spherocytes in the blood smear and increased OF. Their ages ranged from 2 months to 12 years.

Thirty-one patients with hemolytic anemias other than HS were included in this study. Ten cases were diagnosed as thalassemia, eight cases as autoimmune hemolytic anemia and the diagnosis of hemolysis was not reached in 12 cases.

Seventy-four healthy individuals with normal hematological parameters and red cell morphology served as normal controls.

All patients did not receive blood transfusions 1 month before their red cell analyses. All blood samples were anticoagulated with EDTA and stored at 4°C. Flow cytometric analysis of intact red cells was performed within 24 h after blood collection.

This study was approved by a local ethics committee at Alexandria University Hospitals, Egypt.

EMA labeling and flow cytometry

The method described by Tytherleigh et al. [8] was followed. In brief, RBCs were washed twice with PBS, pH 7.4. The supernatant was discarded and 5 μl of red cell pellet were incubated with 25 μl of EMA (1 mg/ml PBS) (Sigma-Aldrich Inc., St Louis, Missouri, USA) for 1 h at room temperature in the dark, followed by centrifugation for 5-10 s in a microcentrifuge, and then the supernatant (containing unbound dye) was removed. The labeled red cells were washed three times with 500 μl of PBS-bovine serum albumin (BSA) solution (0.5% BSA in PBS) [8],[9] .

Red blood cells were suspended in 0.5 ml of PBS-BSA solution. About 100 μl of the cell suspension was transferred into a plastic flow cytometer tube and 1.4 ml of wash solution was added [8],[9] . The median fluorescence intensity (MFI) was determined for 15 000 events in the FL1 channel of a Becton Dickinson FACSCalibur flow cytometer equipped with Cell Quest software (USA) and it was compared with that of normal controls. The mean of the MFI and the range of the MFI for the normal controls were reported. The ratio of MFI of the patient to the range for the normal control samples should be calculated and multiplied by 100 to obtain MFI%.


  Results Top


In the present study, the sex distribution showed no significant difference between HS cases, normal controls and other hemolytic anemia cases (P > 0.05).

Family history showed a significant difference between HS cases and other hemolytic anemias (P < 0.001), with 74.3% of HS having a positive family history and 5.7% being positive for consanguinity; however, there was no significant difference between HS and other hemolytic anemia cases in terms of their symptoms (pallor and jaundice) and blood transfusion requirements (P > 0.05).

Splenomegaly showed a significant difference between HS cases and other hemolytic anemia cases (P < 0.001). About 97% of our HS cases had splenomegaly, whereas 23.5% were splenectomized. About 17% of other hemolytic anemia cases had splenomegaly and were nonsplenectomized.

The presence of spherocytes in the peripheral blood film showed a significant difference between HS cases and those with other hemolytic anemia cases (94.3 and 22%, respectively) (P < 0.001).

The Hb level was significantly higher in HS cases (8.94 ± 1.64) than in cases having other hemolytic anemias (8.01 ± 1.57; P = 0.022). Also, the MCHC level was significantly higher in HS (33.57 ± 2.04) than in cases having other hemolytic anemias (76.33 ± 11.40; P = 0.735). The reticulocyte count in HS cases was elevated, with a mean of 3.74 ± 2.62, but there was no significant difference between HS cases and those with other hemolytic anemias. According to mean cell volume (MCV), mean corpuscular volume (MCH) and red cell distribution width (RDW), there was no significant difference between HS cases and those having other hemolytic anemias.

According to the type of anemia, 51.4% of our HS cases had normocytic anemia, 45.7% had microcytic anemia, whereas the remaining 3.9% of our cases had macrocytic anemia. This is compared with 67.7% of other hemolytic anemia cases had normocytic anemia, 29% had microcytic anemia while 3.2% had macrocytic anemia.

In all HS cases, the OF test was positive (100%), wherein 80% were positive in the fresh test and 20% were negative, whereas after a 24-h incubation, all cases became positive. However, in other hemolytic anemia cases, the test was negative (100%).

The EMA MFI was significantly lower in HS cases (150.93 ± 18.42) than in normal controls (207.37 ± 19.45) and in other hemolytic anemia cases (207.37 ± 19.45; P < 0.001).

Also, the MFI% and the ratio of the MFI of a patient/the MFI of a normal control was significantly lower in HS cases than in other hemolytic anemia cases (P < 0.001) ([Table 1]).
Table 1 Comparing HS cases with other hemolytic anemias according to the MFI, the ratio of MFI of PT over control and the MFI%

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MFI is considered 'excellent' at diagnosing HS: the area under the receiver operating characteristic curve = 0.988, P < 0.001, 95% confidence interval (CI) = 0.92-1.00. Using the Youden index, an MFI of 170 unit or less was determined as the cut-off value with the highest diagnostic performance. At a cut-off value of 170 unit or less, the sensitivity was 91.43% with the 95% CI ranging from 76.9 to 98.2% and a specificity of 100% (95% CI = 88.8-100%).

MFI% is considered 'excellent' at diagnosing HS: the area under the receiver operating characteristic curve 0 =1.00, P < 0.001, 95% CI = (0.95-1.00). Using the Youden index, an MFI% of 87 unit or less was determined as the cut-off value with the highest diagnostic performance. At a cut-off value of 87 units or less, the sensitivity was 100% with the 95% CI ranging from 90.0 to 100% and a specificity of 100% (95% CI = 88.8-100%) ([Figure 1] and [Figure 2]).
Figure 1 ROC curve analysis of MFI. MFI, mean fluorescence intensity; ROC, receiver operating characteristic curve.

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Figure 2 ROC curve analysis of MFI%. MFI, mean fluorescence intensity; ROC, receiver operating characteristic curve.

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  Discussion Top


The diagnosis of HS is generally based on a peripheral smear examination for the presence of spherocytes in a patient with anemia, splenomegaly, hyperbilirubinemia and increased OF. However, these methods are time consuming and labor intensive and have poor specificity and sensitivity, resulting in milder and atypical cases of HS often being missed [7] . A flow cytometry-based analysis by measuring the EMA fluorescence intensity of labeled red cells has been developed.

In the present study, HS cases have a significantly higher positive family history in 74.3% of the cases. These results are similar to the study by Bolton-Maggs et al. [10] and Da Costa [1] , who found that most cases (75%) had a family history of HS.

The Hb level was significantly higher in HS cases than in cases having other hemolytic anemias, which indicates that most HS cases have mild-to-moderate anemia. This is in accordance with the study by Bianchi [11] , who reported lower Hb levels in HS cases.

Regarding the type of anemia, 51.4% of the HS patients had normocytic anemia, and 45.7% had microcytic anemia. This decrease in MCV, especially in severe forms of HS, is due to significant decreases in the spectrin content of the red cell membrane, and the presence of associated iron-deficiency anemia may also explain the low MCV in some cases. One case had severe hemolysis and an elevated reticulocyte count and macrocytic anemia. Da Costa et al. [1] reported that the MCV was also decreased to a variable extent depending on the severity of anemia, which is applicable to the findings in the present study.

Regarding the MCHC level, we found that it was significantly higher in HS (33.57 ± 2.04) than in cases having other hemolytic anemias. In contrast, Da Costa et al. [1] stated that MCHC values were more than 36 g/dl and Eberle et al. [12] reported a high MCHC (35.67 ± 1.33 g/dl) in the HS cases in his study. This higher MCHC level compared with the present study might be explained by the low MCV in some of our cases.

Among the diagnostic methods considered, OF test was carried out and it was found that 20% of HS patients were negative in the fresh test, whereas after a 24-h incubation, all cases became positive. These findings prove the importance of performing an OF test both fresh and after incubation to improve the sensitivity of the test. However, hemolysis might be sometimes masked by the presence of associated iron-deficiency anemia and in mild compensated cases. This indicates the fact that the OF test alone cannot be used to exclude HS because iron deficiency is common among Egyptian children [13] . This is in accordance with the study by Bianchi [11] who found that the OF test failed to identify nearly a quarter of the patients with HS in the fresh test, whereas after a 24-h incubation, the sensitivity of the test improved to 81%.

In the present series, the EMA-binding test was performed and MFI was measured, aiming to differentiate HS cases from other hemolytic anemias. To compare the results, two formats were used: the MFI reading and the MFI%.

An MFI% of 87 unit or less was determined as the cut-off value with the highest diagnostic performance. The EMA cut-off value or the optimum fluorescence decrease that discriminates HS patients from normal controls was equal to or more than 13%.

According to Bianchi et al. [14] the EMA or the optimum fluorescence decrease that discriminates HS patients from controls was 11%. In the Mayeur Rousse et al. [15] study, it was estimated as 10%, and from other hemolytic diseases 14%. Mackiewicz et al. [16] and Girodon et al. [17] calculated the cutoff to be 21% with a 'gray-zone' between 21 and 16%. Park et al. [18] estimated a cutoff of 10.6%.

According to King et al. [19] , the test was considered positive when there was a decrease in the flouresence ratio by greater than 21%, whereas a value of 16% was considered negative. Values between 16 and 21% were considered indeterminate. According to Crisp et al. [20] the estimated cut-off points for EMA-FC were 17%.

In the present study, an MFI of 170 unit or less was determined as the cut-off value with the highest diagnostic performance. Values between 173 and 189 were considered indeterminate. The use of MFI has the advantage of identifying minor differences in borderline normal results, which could be of use in diagnosing mild cases. In contrast, it is impossible to compare results between different labs and different flow cytometery analyzers.

Tachavanich et al. [7] estimated that the optimum cutoff was 91.5 MCF units. In contrast, Stoya et al. [21] suggested an MFI of 400.0 units as the threshold value identified by logistic regression. Also, Kar et al. [22] found that the MFI of confirmed HS cases was 7949.3 (SD 1304.1).

This proves the fact that it is difficult to compare the results between hematology centres when using MFI without calculating the MFI%.

From the present study, we found that the EMA-binding test is easy to use, and the test results are available for reporting in 2-3 h. When HS is suspected in a neonate, the EMA-binding test is the best screening test in neonates as the morphology can be confusing and neonatal red blood cells are more osmotically resistant than adult cells, and so the OF test is less reliable for the diagnosis of this disease.

The test is unaffected by a decrease in the size of red cells that do not have any known cytoskeletal protein deficiency (e.g. red cells from an iron-deficient patient with a lower MCV).

We conclude that the EMA-binding test can be used as the first screening test for HS, provided the red cell morphology and the family history are also taken into account.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Park SH, Park CJ, Lee BR, Kim YJ, Cho YU, Jang S, et al. Screening for hereditary spherocytosis: ema binding test and flow cytometric osmotic fragility test are recommended, but cryohemolysis test is not recommended. Blood 2013; 122 :3425.  Back to cited text no. 18
    
19.
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20.
Crisp RL, Solari L, Vota D, Garcia E, Miguez G, Chamorro ME, et al. A prospective study to assess the predictive value for hereditary spherocytosis using five laboratory tests (cryohemolysis test, eosin-5´-maleimide flow cytometry, osmotic fragility test, autohemolysis test, and SDS-PAGE) on 50 hereditary spherocytosis families in Argentina. Ann Hematol 2011; 90 :625-634.  Back to cited text no. 20
    
21.
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