Analysis of Hereditary Elliptocytosis with Decreased Binding of Eosin-5-maleimide to Red Blood Cells

Abstract

Flow cytometric test for analyzing the eosin-5-maleimide (EMA) binding to red blood cells has been believed to be a specific method for diagnosing hereditary spherocytosis (HS). However, it has been reported that diseases other than HS, such as hereditary pyropoikilocytosis (HPP) and Southeast Asian ovalocytosis (SAO), which are forms in the category of hereditary elliptocytosis (HE), show decreased EMA binding to red blood cells. We analyzed EMA binding to red blood cells in 101 healthy control subjects and 42 HS patients and obtained a mean channel fluorescence (MCF) cut-off value of 36.4 (sensitivity 0.97, specificity 0.95). Using this method, we also analyzed 12 HE patients. Among them, four HE patients showed the MCF at or below the cut-off value. It indicates that some HE patients have decreased EMA binding to red blood cells. Two of these four HE patients were classified as common HE, and two were spherocytic HE with reduced spectrin. This study demonstrates that, in addition to patients with HPP or SAO, some HE patients have decreased EMA binding to red blood cells.

Figure 1

EMA binding in healthy subjects, HS patients, and HE patients. MCF in healthy subjects = 39.2 ± 1.6 (n = 101), MCF in HS patients = 28.8 ± 3.8 (n = 42), and MCF in HE patients = 35.6 ± 3.3 (n = 12) (MCF cut-off value: 36.4).

Figure 2

SEM images and results of SDS-PAGE gels of HE patients with decreased EMA binding to red blood cells. (a) SEM image and SDS-PAGE of Family A (case 1, case 2). SDS-PAGE: proband (case 1): spectrin 14.2% reduction, mother (case 2): spectrin 14.5% reduction. (b) SEM image and SDS-PAGE of case 3. SEM: ovalocytic type and rod-shaped type were observed. SDS-PAGE: no membrane protein abnormality detected. (c) SEM image and SDS-PAGE of case 4. SEM: many rod-shaped types were observed. SDS-PAGE: no membrane protein abnormality detected.

Figure 3

Association between the presence/absence of decreased EMA binding and elliptocyte development in HE patients. (a) Association between the presence/absence of decreased EMA binding and the percentage of rod-shaped type in HE patients. The percentage of rod-shaped type in 4 HE patients with decreased EMA binding = 18.0 ± 12.7%. The percentage of rod-shaped type in 8 HE patients without decreased EMA binding = 15.3 ± 13.2%. Rod-shaped type: RBC long diameter/short diameter ≥2. (b) Association between the presence/absence of decreased EMA binding and the percentage of ovalocytic type in HE patients. The percentage of the ovalocytic type in 4 HE patients with decreased EMA binding = 72.2 ± 6.6%. The percentage of the ovalocytic type in 8 HE patients without decreased EMA binding = 69.4 ± 10.4%. Ovalocytic type: RBC long diameter/short diameter <2.