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العنوان
Usefulness of Eosin-5’-Maleimide (EMA) Dye for the Diagnosis of Hereditary Spherocytosis /
المؤلف
Hassaballah,Dina Muhammed Saied.
هيئة الاعداد
باحث / Dina Muhammed Saied Hassaballah
مشرف / Azza Sadek Eldanasoury
مشرف / Deena Mohamed Mohamed Habashy
مشرف / Iman Ahmed Ragab
تاريخ النشر
2019.
عدد الصفحات
159p.:
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
أمراض الدم
تاريخ الإجازة
1/1/2019
مكان الإجازة
جامعة عين شمس - كلية الطب - الباثولوجيا الاكلينيكية
الفهرس
Only 14 pages are availabe for public view

from 159

from 159

Abstract

Hereditary spherocytosis (HS) is a common congenital hemolytic anemia. The typical laboratory hallmark of HS is the presence of spherocytes in peripheral blood film. HS is characterized by minimal or no anemia, an increased mean corpuscular hemoglobin concentration (MCHC), spherocytes on the peripheral blood smear and abnormal results on the osmotic fragility test (OFT).
While OFT has long been considered the gold standard for diagnosing of HS, this test requires a large volume of blood and an incubation period of 24 hours and it lack specificity for diagnosis of HS.
A flow cytometric method, based on the fluorescence of erythrocytes after incubation with eosin-5′-maleimide (EMA) dye, had been described for the diagnosis of HS. Chemical studies showed that this fluorescence was mainly attributable to the band 3 protein (80%) and, to a lesser degree, to proteins associated with the Rh blood group protein complex in the erythrocyte membrane.
Absent or decreased expression of RBC membrane proteins found in HS causes a disruption of the cytoskeleton network and reduces normal expression of band 3 protein at the erythrocyte membrane. This results in a reduced binding of EMA to band 3 protein and thus its fluorescence emission.
The aim of this our study was to evaluate the ability EMA dye binding flow cytometry in diagnosis of HS and assess the sensitivity and specificity of this test to distinguish HS patients from patients having anemia due to other causes to be applied in routine diagnostic workup.
This study was conducted on 52 patients with chronic anemia, 21 already diagnosed as HS (group I) based on clinical features (anemia, jaundice and splenomegaly), family history, spherocytes in blood smear and increased osmotic fragility. 31 patients having anemia due to other causes (group II). 10 cases diagnosed as autoimmune hemolytic anemia (AIHA) (group IIa), 11 cases as iron deficiency anemia (IDA) (group IIb) and 10 cases with beta thalassemia intermedia (BTI) (group IIc).
54 healthy subjects were recruited as a control group with normal hematological parameters and red cell morphology (group III).
All patients didn‟t receive blood transfusion in the last month prior to their red cell analyses. At least two normal control samples were collected with each case under the same condition. All blood samples were anticoagulated with EDTA and kept at 4ºC.
Patients‟ RBCs were incubated with EMA, the median fluorescence intensity (MFI) of each case and its controls were measured and %reduction in EMA fluorescence had been calculated in all studied patients.
% EMA reduction =
[(mean of MFI of 6 controls)ˍ (MFI of the patient)]×100
mean of MFI of the 6 controls
The patients‟ results had been compared to the mean and range of the control samples and to other patient‟s samples as well. MFI had shown significantly lower values in HS patients (group I) (3.78±0.48) than IDA patients (group IIa) (5.56±0.35), AIHA patients (group IIb) (5.7±0.27), BTI patients (group IIc) (6.01±0.63) and control group (group III) (5.3±0.43). The % reduction in EMA fluorescence proved to be significantly higher in HS patient (group I) (29.88±8.27) than in IDA patients (group IIa) (1.13±6.18), AIHA patients (-6.14±10.44) and BTI patients (group IIc) (-5.1±10.07). Multi regression analysis had been used to check the most sensitive predictors for diagnosis of HS, both EMA MFI and the % decrease in EMA fluorescence proved to be the best sensitive predictive values for discriminating HS patients, however the higher F-ratio in EMA MFI (F=25.222) than in % decrease in fluorescence intensity (F=19.866) showed that the MFI is even better than % decrease in fluorescence in diagnosis of HS.
A receiver operating characteristic (ROC) curve was used to elicit the best cutoff for EMA MFI and % reduction in EMA fluorescence with the best sensitivity and specificity to differentiate HS from control group and from other different causes of anemia. In the present study, the sensitivity and specificity achieved 100% at a cutoff for MFI was at 4.77 and 11.9% reduction in EMA fluorescence intensity.
In conclusion, flow cytometric analysis using the EMA dye is a sensitive, specific, cost effective, highly reproducible, less labor intensive and far more sensitive in diagnosis of HS test when compared to conventional methods, which can be used as a simple and practical test for HS screening. The collaboration between clinicians and laboratories is very important and should take into account the family history and the exclusion of other causes of secondary spherocytosis.
Though flow cytometery may not be available in under resourced facilities, our study has proved without doubt that whenever a flow cytometer is available, this method of testing is helpful to deliver accurate reports to the patients and clinicians at a much shorter time. However, further investigation by confirmatory tests are recommended.