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Advances in the laboratory diagnosis of hereditary spherocytosis

par Lazarova, Elena
Président du jury Corraza, Francis
Promoteur Gulbis, Béatrice
Publication Non publié, 2021-10-26

Thèse de doctorat

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Résumé :

Establishing the diagnosis in a patient with hereditary hemolytic anemia is a complex and laborious procedure that involves a comprehensive evaluation of clinical and laboratory features. Because the group of associated pathologies is very heterogeneous with regard to underlying cause, disease severity, age of onset and clinical course, ascertaining the correct diagnosis can be challenging. Thereby, even cases of typical clinical and biological presentation of hereditary spherocytosis (HS), the main cause of hereditary hemolytic anemia in Northern Europe, need confirmation by specialized screening and diagnostic tests. Unfortunately, with reported sensitivities of 53%-95%, none of those tests alone is able to identify all cases of HS, in particular, mild and moderate forms of HS, and the use of at least two screening tests was suggested. Therefore, the general objective of this research was to investigate and introduce into clinical practice new screening and diagnostic tools for hereditary spherocytosis, in order to improve the laboratory diagnostic algorithm. Based on the pathophysiology of HS, it has been hypothesized that automated reticulocyte parameters could be of great interest as screening tests especially if able to demonstrate the presence of small dehydrated reticulocytes coexistent with spherocytes. Thereby, we were first interested in investigating the new automated reticulocyte parameters as a screening tool for HS. We analyzed the diagnostic performances of these parameters, estimated their efficiency in differentiating HS from other conditions that affect erythropoiesis, and introduced them into a new screening algorithm for HS as a highly efficient first line test. Secondly, we were interested in the osmotic gradient ektacytometry, a diagnostic tool for HS and other membrane pathologies, already known for several decades. However, this method remained with very limited use only in highly specialized laboratories because of the complexity of the equipment. Lately, a next-generation device has been introduced: the laser-assisted optical rotational cell analyzer (LoRRca) which generates typical osmoscan curves for various RBC membrane pathologies as previous ektacytometers did. Thereafter, we were specifically interested in the diagnostic accuracy of the various parameters available from the osmoscan curve and their clinical application for HS diagnosis. We established strict diagnostic cut-off values for those parameters, and proposed introducing the osmoscan into the diagnostic workflow of RBC membrane disorders as an efficient intermediate step between screening tests and confirmatory protein deficiency tests or DNA-based methods in a reference laboratory. Finally, although considered mainly as pediatric disease, HS could be also initially diagnosed in adults during family studies or accidentally found enlarged spleen or gallstones. However, older patients are often affected by common medical conditions like diabetes or dyslipidemia, known to be characterized, inter alia, by hemorheological alterations and decreased RBC deformability, which are typical pathophysiological features of HS. Therefore, we investigated and demonstrated that diabetes or dyslipidemia do not interfere with the laboratory methods for HS diagnosis. Our work was acknowledged as significant in its field for HS screening and differential diagnosis and it was included in the last guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. Together, with its larger use and recent wider clinical applications, LoRRca ektacytometer, commercialized as laboratory equipment, gathered popularity and data from several clinical studies have been collected. Furthermore, method standardization and inter-laboratory result comparison became possible and the osmoscan added value as an intermediate step in the laboratory diagnosis of HS versus other hereditary RBC membrane disorders has been confirmed. Establishing strict diagnostic cut-offs for each of the tests and parameters, introducing specific results expression and investigating interferences with other pathologies, allowed us to position these new methodologies into the good laboratory practice of the 21st century.