Contributions
Abstract: PB1983
Type: Publication Only
Background
Measuring the fluorescence intensity of eosin-5-maleimide (EMA) labelled red cells has been shown to be an effective screening for the detection of hereditary spherocytosis (HS), particularly in combination with acidified glicerol test or osmotic fragility test. EMA predominantly binds covalently to the ε-NH2 group of the lysine-430 in the band 3 protein of the red cell cytoskeleton. The red cells of patients with hereditary spherocytosis have a lower EMA mean channel fluorescence intensity (MFI) than normals and other haemolytic anaemias.
Reference ranges according to local population must be calculated. In our experience, absolute values of MFI undergo small changes on each reagent preparation, so the difference regarding a mean of many controls acquire more signification. Reference ranges of neonatal population have not been well established.
Aims
The aim of this study was to establish the reference ranges of EMA-test in the neonatal and paediatric population, and to review the current values expected in the adult population
Methods
Twenty-three venous blood samples from 21 newborn patients (10 women and 11 men), aged from 0 to 30 days (n=19), 60 days (n=1) and 90 days (n=1) were taken from patients attended in the emergency. Only 4 of the cases were anaemic.
Moreover, 33 adult patients with an EMA test performed were reviewed. Other tests were considered, such as acidified glycerol test, osmotic fragility test and RBC membrane protein quantification by SDS-PAGE electrophoresis, to establish a final diagnosis of HS.
Five microliters of whole blood from each sample was washed with 0.9 % NaCl solution and incubated in darkness at room temperature for 1 h with 25 μL of EMA dye (0.5 mg/mL, in phosphate-buffered saline, PBS), with intermittent mixing. Stained red blood cells were washed three times using 0.5 %/FBS/PBS and centrifuged after each wash. Cells were suspended in 500 μL of 0.5 % FBS/PBS solution, and 100 μL of labelled cell suspension was diluted in 1.4 mL of FBS/PBS. Flow cytometric analysis was carried out using Cytomics FC 500 flow cytometer (Beckman Coulter, Fullerton, CA, USA). Blood samples from six blood donors were used as normal controls for each assay.
The reference interval was calculated according to the CLSI guidelines, C28-A3 and a robust method was used.
Results
Reference 95% interval for newborn patients was -33.5 to 17.9, ranging from a very negative value to an almost normal adult value.
Reference intervals for adult individuals were changed from those recommended in the literature (difference > 16% suspected HS, and difference >21% compatible with hereditary spherocytosis) to 11% and 16%, respectively. Details of each case will be provided.
Conclusion
Reference interval for newborn patients has a wide range. Studies on HS newborns must be conducted to establish the range of suspected HS.
Adult reference interval for older children and adults should be calculated for each local population, as it may differ significantly from the original recommendations.
Session topic: 29. Enzymopathies, membranopathies and other anemias
Keyword(s): Hereditary spherocytosis
Abstract: PB1983
Type: Publication Only
Background
Measuring the fluorescence intensity of eosin-5-maleimide (EMA) labelled red cells has been shown to be an effective screening for the detection of hereditary spherocytosis (HS), particularly in combination with acidified glicerol test or osmotic fragility test. EMA predominantly binds covalently to the ε-NH2 group of the lysine-430 in the band 3 protein of the red cell cytoskeleton. The red cells of patients with hereditary spherocytosis have a lower EMA mean channel fluorescence intensity (MFI) than normals and other haemolytic anaemias.
Reference ranges according to local population must be calculated. In our experience, absolute values of MFI undergo small changes on each reagent preparation, so the difference regarding a mean of many controls acquire more signification. Reference ranges of neonatal population have not been well established.
Aims
The aim of this study was to establish the reference ranges of EMA-test in the neonatal and paediatric population, and to review the current values expected in the adult population
Methods
Twenty-three venous blood samples from 21 newborn patients (10 women and 11 men), aged from 0 to 30 days (n=19), 60 days (n=1) and 90 days (n=1) were taken from patients attended in the emergency. Only 4 of the cases were anaemic.
Moreover, 33 adult patients with an EMA test performed were reviewed. Other tests were considered, such as acidified glycerol test, osmotic fragility test and RBC membrane protein quantification by SDS-PAGE electrophoresis, to establish a final diagnosis of HS.
Five microliters of whole blood from each sample was washed with 0.9 % NaCl solution and incubated in darkness at room temperature for 1 h with 25 μL of EMA dye (0.5 mg/mL, in phosphate-buffered saline, PBS), with intermittent mixing. Stained red blood cells were washed three times using 0.5 %/FBS/PBS and centrifuged after each wash. Cells were suspended in 500 μL of 0.5 % FBS/PBS solution, and 100 μL of labelled cell suspension was diluted in 1.4 mL of FBS/PBS. Flow cytometric analysis was carried out using Cytomics FC 500 flow cytometer (Beckman Coulter, Fullerton, CA, USA). Blood samples from six blood donors were used as normal controls for each assay.
The reference interval was calculated according to the CLSI guidelines, C28-A3 and a robust method was used.
Results
Reference 95% interval for newborn patients was -33.5 to 17.9, ranging from a very negative value to an almost normal adult value.
Reference intervals for adult individuals were changed from those recommended in the literature (difference > 16% suspected HS, and difference >21% compatible with hereditary spherocytosis) to 11% and 16%, respectively. Details of each case will be provided.
Conclusion
Reference interval for newborn patients has a wide range. Studies on HS newborns must be conducted to establish the range of suspected HS.
Adult reference interval for older children and adults should be calculated for each local population, as it may differ significantly from the original recommendations.
Session topic: 29. Enzymopathies, membranopathies and other anemias
Keyword(s): Hereditary spherocytosis