IDENTIFICATION AND VERIFICATION OF HEREDITARY SPHEROCYTOSIS BY MEANS OF LABORATORY DIAGNOSIS

Aim. Hereditary spherocytosis (HS) is the most commonly encountered erythrocyte membranopathy. Frequency of occurrence of the disease makes one case per 2000−5000 newborns. Hereditary spherocytosis often causes a complex of clinical signs, including hemolytic crises in patients. At the same time many patients have asymptomatic HS. Differential diagnosis of HS is quite complex and in modern workload conditions the clinical doctors need a simpler diagnostics procedure. Patients and methods. Participants included 13 adults with verified hereditary spheroсytosis and 42 children with identified hereditary spherocytosis, 311 adults without hematological disorders, 42 children without hematological disorders. Verification of hereditary spherocytosis diagnosis was carried out using flow cytometry test (eosine-5 maleimid-binding), Deich`s method of determination of erythrocyte osmotic resistance and Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis. In this study we have assessed diagnostic value of hematological parameters provided by the hematological analyzer Beckman Coulter Cellular Analysis System DxH800 for identifying the degree of erythropoiesis disorder in patients with hereditary spherocytosis at the stage of reticulocytes maturation. According to our data, the ratio RET/IRF and calculated parameter MCV-MSCV can be used as the screening tests for hereditary spherocytosis. Results. Evaluation of the erythrocytes and reticulocytes parameters at the hematological analyzer identified the significant difference in estimate index MCV-MSCV (p < 0,0001, sensitivity 100%, specificity 100%, area under the ROC-curve 1,0) and RET/IRF (p < 0,0001, sensitivity 96,3%, specificity 94,1, area under the ROC-curve 0,97) between group of patients with HS and control group. We also evaluated the usability of eosine-5 maleimide binding in flow cytometry for verification of this membranopathy. For unify the test results we offer to use estimate indicator S (sample`s MFI / control`s mean MFI), cut level for positive cases of hereditary spherocytosis S < 0,84 (p = 0,0001, sensitivity 98,2%, specificity 99,2%, area under the ROC-curve 0,99). Conclusion. We recommend the hematological analyzer evaluation as the screening option for the identification of HS in patients and determine the estimated parameters for the values of the patients MCV-MSCV and RET/IRF. The most informative verifying test to prove hereditary spherocytosis is the flow cytometry test using eosine-5 maleimid. It is the laboratory test that proves a high degree of sensitivity and specificity for hereditary spherocytosis. Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis of red blood cells membranes proteins is useful for specify molecular deficiency in each hereditary spherocytosis case.

1. Rukavitsyn O.A., Pavlov A.D. Anemii. Pod red. O.A. Rukavitsyna, A.D. Pavlova [Anemia. Edited by O.A. Rukavitsyn, A.D. Pavlov]. St. Petersburg, Detstvo-Press, 2011. P. 67−68.

2. Mullier F., Lainey E., Fenneteua O., Da Costa L., Schillinger F., Bailly N., Cornet Y., Chatelian C., Dogne J.-M., Chatelian B. Additional erythrocytic and reticulocytic parameters helpful for diagnosis of hereditary spherocytosis: results of a multicentre study. Annals of hematology. July 2011; 90: 759−768.

3. Guseva S.A., Voznyuk V.P., Dubkova A.G. Anemii: printsipy diagnostiki i lecheniya. Pod red. S.A. Gusevoi [Anemia: principles of diagnosis and treatment. Edited by S.A. Guseva]. Kiev, 1999. P. 74−75.

4. Fourcade Ch., Jary L., Belaouni H. Reticulocyte Analisys Provided by the Coulter GEN S significance and interpretation in regenerative and nonregenerative hematologic conditions. Laboratory hematology. 1999; 5: I−xx.

5. Cniron M., Cynober T., Mielot F., Тchernia G., Croisille L. The GENs: a fortuitous finding of a routine screening test for hereditary spherocytosis. Hematol Cell Ther. 1999; 41: 113−116.

6. Agre P., Orringer E.P., Bennett V. Deficient red-cell spectrin in severe, recessivelly inherited spherocytosis. N Engl J Med. 1982; 306: 1155−61.

7. King M.J., Zanella A. Hereditary red cell membrane disorders and laboratory diagnostic testing. Int J Lab Hematol. 2013 Jun; 35 (3): 237−43.

8. Girodon F., Garcon L., Bergion E., Largier M., Delaunay J., Feneant-Thibault M., Maynadie M., Couillaud G., Moreira S., Cynober T. Usefulnes of the eosin-5 maleimide cytometric method as a first-line screening test for the diagnosis of hereditary spherocytosis: Comparison with ektacytometry and electrophoresis. Br J Haematol. 2008; 140: 468−70.

9. Kar R., Mishra P., Pati H.P. Evalution of eosin-5-maleimide flow cytometric test in diagnosis of hereditary spherocytosis. Int Jnl Lab Hem. 2010; 32: 8−16.

10. Prokhorova Yu.A., Zueva E.E., Sokolova N.E. Application of flow cytometry in diagnosis of hereditary spherocytosis (binding test eosin maleimide-5). Klinicheskaya laboratornaya diagnostika – Clinical laboratory diagnostics. 2012; 7: 31−5.

11. Doherty G.J., McMahon H.T. Mediation, modulation and consequences of membrane-cytoskeleton interactions. Annual Review of Biophisics. 2008; 37: 73.

12. King M.J., Jepson M.A., Guest A., Mushens R. Detection of hereditary pyropoikilocytosis by the eosin-5 maleimide(EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins. Int. Journal of laboratory Hematology. 2011; 33: 205−211.

13. Kedar P.S., Colah R.B., Kulkarni S., Ghosh K., Mohanty D. Expirience with eosin-5-maleimide as a diagnostic tool for red cell membrane cytoskeleton disorders. Clin Lab Haematol. 2003; 25: 373−6.

14. Nazarenko G.I., Kishkun A.A. Klinicheskaya otsenka rezul'tatov laboratornykh issledovanii [Clinical evaluation of laboratory results]. Moscow, Izd. Meditsina, 2005. P. 39−41.

15. Baranov A.A., Semikina E.L., Mel'nichuk O.S., Gordeeva O.B., Namazova-Baranova L.S., Morozova N.A., Kozhevnikova O.V., Gevorkyan A.K., Mayanskii N.A. Indicators retikulotsitarnyh indices in healthy children. Voprosy diagnostiki v pediatrii – Diagnostics in pediatrics. 2010; 4 (2): 19−21.

16. Lazarova E., Pradier O., Cotton F., Gulbis B. Automated reticulocytes parameters for hereditary spherocytosis screening. Ann Hematol. 2014 Jun; 10: 58−69.

17. Broseus J., Visomblain B., Guy J., Maynadie M., Girodon F. Evaluation of mean sphere cospuscular volume for predicting hereditary spherocytosis. Int J Lab Hem. 2010; 32 (5): 521−522.