Joan-Lluis Vives Corrons1, Elena Krishnevskaya1, Ines Hernandez Fernandez2, Salvador Payan Pernia3

1–Red Cell Pathology and Hematopoietic Disorders (Rare Anemias Unit), Josep Carreras Institute for Leukaemia Research , Badalona (Barcelona);  2–Hematology Laboratory Department, Hospital Universitari Germans Trias i Pujol , Badalona (Barcelona), Spain,  3-Hospital Universitario Virgen del Rocío, Seville, Spain

It has been demonstrated that the combination of t-NGS and RBC deformability measured by OGE using a laser-assisted optical rotational ektacytometer (LoRRca) is the most powerful contribution to the clear differentiation between HS and HE. The possibility that in HPP, RBCs appear to have a significantly  decreased  ability  to  maintain  deformability  in  the  face  of  hypertonicity  makes  the classical  trapezoidal  profile  of  HE  is  less  evident.  Our  objective  is  to  provide  evidence  that  in Hereditary  Pyropoikilocytosis  (HPP),  the  implication  of  a  canonical  splicing  site,  may  explain,  not only the marked defect of spectrin chains tetramerisation and elliptocytosis, but also the weakness of skeleton attachment to the  lipid bilayer and the loss of RBC membrane with microvesiculation and formation of spherocytes as in HS.
RBC  morphology  is  a  key  diagnostic  feature  for  hereditary  spherocytosis  (HS)  and  hereditary elliptocytosis (HE). However, this phenotypic diagnosis may not be readily available in patients with high reticulocyte counts and/or requiring frequent transfusions and does not allow to predict the disease clinical course or its severity. However, RBC deformability measured by osmotic gradient ektacytometry  (OGE)  allows  a  clear  differentiation  between  HS  and  HE,  where  the  truncated osmoscan curve  reflects the  inability of the already elliptical cells to deform further under shear stress. In the severe clinical form of HE called Hereditary Pyropoikilocytosis (HPP), the RBCs appear to have a significantly decreased ability to maintain deformability in the face of hypertonicity and the classical trapezoidal profile of HE is less evident making it indistinguishable from the HS profile.  Here,  two  unrelated  patients  with  severe  HHA  due  to  HPP  and  HS,  are  described  with  the  joint inheritance of a complex set of five variants, all located in the SPTA1 gene. Two of these variants are novel and not described before, one  is a microdeletion that removes  the  entire SPTA1 gene, and two are the known low‐expression polymorphic alleles:  α-LELY and α-LEPRA.  Both patients  exhibit the  same  HS  osmoscan profile  suggesting  that  in HPP  patient  with marked spherocytosis, the interactions between the two SPTA1 gene variants may led , in addition to the elongation effect (elliptocytes) to a loss of membrane stability and microvesiculation (spherocytes) (1,2,3)    Accordingly  the  RBCs  appear  to  have  a  significantly  decreased  ability  to  maintain deformability in hypotonic conditions and due  to this,  the  trapezoidal profile of HE may become indistinguishable from HS, highlighting the importance of OGE (ektacytometry) for the differential diagnosis between HS and HPP in patients with no family history of RBC cytoskeleton. 
The  diagnosis  of  Hereditary  Hemolytic  Anemia  (HHA)  was  performed  via  a  step-wiseprocess including  RBC  morphology,  Hb  electrophoresis,  and  measurement  of  common  RBC  enzyme activities.  RBC  deformability  and  other  rheological  parameters  were  studied  by  OGE,  using  the osmoscan  module  of  the  Laser-assisted  Optical  Rotational  DeformabilityCell  Analyser  (LoRRca; MaxSis. RR Mechatronics) as previously described (4). 
(Osmoscan): The  RBCs are  submitted  to an  increasing  osmotic gradient  (80 -550  mOsmol/L)  under a constant shear force (<30 Pa)
Genetic diagnosis of membranopathies was performed by gene capture followed by t –NGS that includes a panel of 35 genes responsible for membranopathies hemoglobinopathies, enzymopathies and congenital dyserythropoietic anemias (CDA) t-NGS and Whole exome sequencing (WES) have been performed usingan Illumina HiSeq2000 device. 
Our study demonstrates that
  1. in HPP, with a high percentage of spherocytes and typical HS osmoscan profile, the interactions between both SPTA1 gene variants, in addition to the loss of shape recovery after elongation (elliptocytes), gives rise to a membrane instability leading to spherocytes which percentage may be depending on the severity of the tetramerisation failure.  
  2. The importance of OGE combined with t-NGS for the diagnosis of patients with RBC morphology characteristic of HPP and clinical phenotype of hereditary hemolytic anemia (HHA) of unknown etiology.
1.Wilmotte R, Marechal J, Delaunay J. Mutation at position -12 of intron 45 (c-->t) plays a prevalent role in the partial skipping of exon 46 from the transcript of allele alphaLELY in erythroid cells. Br J Haematol. 1999; 104 :855-9.  2.Wichterle H. et al. Combination of two mutant alpha spectrin alleles underlies a severe spherocytic hemolytic anemia, The J. Clin.Invest.1996; 98: 2300-2307 3.Gallagher P. G. et al. Aberrant splicing contributes to severe α-spectrin–linked congenital hemolytic anemia //The J. Clin. Invest. 2019; 129: 2878-2887.  4.Llaudet-Planas E; Vives-Corrons JL; Rizzuto V; Gómez-Ramírez P; Sevilla Navarro J; Coll Sibina MT; et al. Osmotic gradient ektacytometry: A valuable screening test for   hereditary spherocytosis and other red blood cell membrane disorders. Int. J. Lab. Hem. 2018; 40: 94-102
The data in this poster was presented at EHA 2021. Published with permission from the Copyright owner.