Evaluation of the Role of Flow Cytometry as a Diagnostic Tool for Hereditary Spherocytosis

Abstract Flow cytometric analysis of eosin-5′-maleimide-labeled red blood cells has been proposed as a method of identifying hereditary spherocytosis (HS). The flow cytometric test measures the fluorescence intensity of intact red cells labelled with the dye eosin-5-maleimide (EMA), which reacts covalently with Lys-430 on the first extra cellular loop of band 3 protein. Patients with HS have reduced fluorescence compared to other patient groups and normal controls. The aim of the present study was to assess the utility of flow cytometry in the diagnosis of hereditary spherocytosis. Fresh peripheral blood from 40 normal controls was collected in Lithium Heparin, stained with the dye EMA and analysed by flow cytometry. The normal range was established as 55–74 channel numbers. On 7 known cases of Hereditary Spherocytosis the MFI range was 35.6–44.6 channel numbers, and therefore samples falling into this range were considered to be positive for a diagnosis of HS by EMA. Equivocal results were defined when the MFI was in the range of 45–54 channel numbers. A total of 98 samples were sent for investigation or exclusion of HS. Clinical problems were grouped into the following categories: peripheral blood spherocytosis with negative Coomb’s test and no family history of HS, positive family history of HS, Neonatal hyperbilirubinaemia, haemolytic anaemia of unknown origin. Indications Coomb’s −ve, Spherocytosis, no FH (n=50) (51%) Positive family history (FH) (n=18) (17%) Neonatal hyperbilirubinemia (NNH) (n=8) (8%) Haemolytic anaemia (HA) (n=16) (17%) Results of EMA Pos Neg Equi Pos Neg Equi Pos Neg Equi Pos Neg Equi Number 19 17 13 9 7 2 2 4 2 0 14 2 Percentage 38 34 26 50 39 11 25 50 25 0 87.5 12.5 Within this cohort the group with FH of HS had the highest positive and least equivocal results. The group with suspected HS (peripheral blood spherocytosis with negative coomb’s test) resulted in positive in 38% and equivocal in 26% cases. The group with NNH had positive results in 25% of cases and the group with HA had no positive results. Equivocal results occurred in 20% of cases suggesting further investigations are required to confirm or exclude HS. The EMA dye method by flow cytometry is a useful test for diagnosing red cell membrane abnormalities due to band 3 defects, but the assay produces a substantial number of equivocal results, and has poor utility in the investigation of haemolytic anaemia of unknown origin.