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Automated reticulocyte parameters for hereditary spherocytosis screening.

 

Abstract

The laboratory diagnosis of hereditary spherocytosis (HS) is based on several screening and confirmatory tests; our algorithm includes clinical features, red blood cells morphology analysis and cryohaemolysis test, and, in case of positive screening, sodium dodecyl sulphate polyacrylamide gel electrophoresis as diagnostic test. Using the UniCell DxH800 (Beckman-Coulter) haematology analyser, we investigated automated reticulocyte parameters as HS screening tool, i.e. mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF) and mean sphered cell volume (MSCV).

A total of 410 samples were screened. Gel electrophoresis was applied to 159 samples positive for the screening tests. A total of 48 patients were diagnosed as HS and 7 were diagnosed as acquired auto-immune haemolytic anaemia (AIHA). Some other 31 anaemic conditions were also studied.

From the ROC curve analysis, both delta (MCV-MSCV) and MRV presented an AUC of 0.98. At the diagnostic cut-off of 100% sensitivity, MRV showed the best specificity of 88% and a positive likelihood ratio of 8.7. The parameters IRF, MRV and MSCV discriminated HS not only from controls and other tested pathologies but also from AIHA contrary to the cryohaemolysis test.

In conclusion, automated reticulocyte parameters might be helpful for haemolytic anaemia diagnostic orientation even for general laboratories. In combination with cryohaemolysis, they ensure an effective and timesaving screening for HS for more specialised laboratories.

PMID: 24912770

 

Supplement:

The last published diagnostic guidelines for hereditary spherocytosis (HS) diagnosis, those of the International Council for Standardization in Haematology International society [1], recommend a preliminary laboratory diagnosis including red cell morphology and full blood count results. In those guidelines, the automated reticulocyte parameters are introduced as being appropriate to differentiate hereditary from acquired haemolytic anaemias.

We studied the usefulness of those parameters as first line laboratory test for HS diagnosis. The value of the parameter MSCV is expected to be higher than that of MCV as a result of cell volume increase after incubation in the hypo-osmolar haemoglobin leaking solution during reticulocyte counting. However, the MSCV value obtained for HS red cells remains lower than that their MCV value because HS red cells are unable to increase further their volume even in a hypo-osmolar medium. Unfortunately, MSCV is a unique parameter of Beckman-Coulter. So we analysed in parallel two other automated parameters, mean reticulocyte volume (MRV) and immature reticulocyte fraction (IRF), and they both proved their efficiency. As MRV and Ret/IRF ratio are available on other haematology automates, contrary to MSCV, we are persuaded that a screening for HS using those parameters could be applied in every laboratory utilising a new generation haematology analyser with flow cytometry based reticulocyte count. Nevertheless, one should be cautious that a local determination of the reference range is important to be conducted.

In conclusion, the introduction of reticulocyte automated parameters in hereditary spherocytosis screening revealed to be clinically relevant. Additionally, we propose this practical screening algorithm for diagnostic orientation of haemolytic anaemia, feasible even for general laboratories. However, we would like to underline, that final HS diagnosis should always be the result of tight collaboration between clinicians and laboratory specialists, considering the family history and excluding other causes of haemolytic anaemia.

 

References

1. King MJ, Garçon L, Hoyer JD, Iolascon A, Picard V, Stewart G, et al. International Council for Standardization in Haematology. ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. Int J Lab Hematol. 2015; 37:304-25