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Erythrocyte Flow Cytometric Analysis in Congenital Dyserythropoietic Anemia Type III-Evaluation of Eosin-5´-Maleimide, CD55, and CD59
Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik.
Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Allmänmedicin.
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2013 (Engelska)Ingår i: Journal of Blood Disorders & Transfusion, ISSN 2155-9864, Vol. 121, nr 23, s. 4791-4799Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Introduction: Flow cytometry with eosin-5´-maleimide (EMA), anti-CD55 and anti-CD59 is commonly used when investigating non-autoimmune hemolytic anemias. Reduced fluorescence of EMA, typically detected in hereditary spherocytosis is also seen in congenital dyserythropoietic anemia type II (CDA II). Reduction of CD55 and CD59 characterizes paroxysmal nocturnal hemoglobinuria (PNH). We studied the flow cytometric profile of EMA, CD55 and CD59 on erythrocytes in congenital dyserythropoietic anemia type III (CDA III). 

Methods: Erythrocytes from 16 CDA III positive individuals, 14 CDA III negative relatives and three normal controls per assay were studied with flow cytometry after EMA staining. Flow cytometry after anti-CD55 and anti- CD59 was performed on erythrocytes from 12 CDA III positive and 7 CDA III negative relatives with one normal control per assay. 

Results: CDA III - erythrocytes exhibited marginally stronger fluorescence after EMA-staining than normal controls. Correlation between EMA fluorescence and erythrocyte volume was confirmed. CDA III subjects did not differ from normal controls concerning CD55 and CD59. 

Conclusion: The results of the present study indicate no abnormality of the erythrocyte membrane in CDA III and show that standard flow cytometry cannot be used to discriminate between CDA III and normal controls. 

Ort, förlag, år, upplaga, sidor
OMICS International , 2013. Vol. 121, nr 23, s. 4791-4799
Nationell ämneskategori
Hematologi
Identifikatorer
URN: urn:nbn:se:umu:diva-117449DOI: 10.4172/2155-9864.1000172OAI: oai:DiVA.org:umu-117449DiVA, id: diva2:907773
Tillgänglig från: 2016-02-29 Skapad: 2016-02-29 Senast uppdaterad: 2021-12-07Bibliografiskt granskad
Ingår i avhandling
1. Congenital Dyserythropoietic Anemia type III (CDA III): diagnostics, genetics and morbidity
Öppna denna publikation i ny flik eller fönster >>Congenital Dyserythropoietic Anemia type III (CDA III): diagnostics, genetics and morbidity
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The Congenital Dyserythropoietic Anemias (CDA) are rare hereditary hemolytic disorders with large bi- to multi-nucleated erythroblasts in the bone marrow. Hemolysis is negative in a direct antiglobulin test (DAT). Based on morphology and clinical picture, three major forms of CDAs, type I, II, and III have been defined. CDA III, dominantly inherited, constitutes the rarest type with a majority of cases belonging to a family in Västerbotten, Sweden. The genetic background of CDA I and CDA II has been linked to mutations in CDAN1 and SEC23B respectively. The mutation of CDA III has been linked to 15q22 in earlier studies.

In this project we have defined the causative genetic lesion in two families with CDA III. The novel mutation KIF23 c.2747C>G (p.P916R) was shown to segregate with CDA III in the Swedish and American CDA III families and was absent in 356 healthy controls. KIF23 encodes mitotic kinesin-like protein 1 (MKLP1), which plays a central role in the last step of cytokinesis. RNAi-based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, resulting in increasing number of bi-nuclear cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23, encoding MKLP1, a conserved mitotic kinesin crucial for cytokinesis.

Flow cytometry with eosin-5´-maleimide (EMA), anti-CD55 and anti-CD59 is commonly used when investigating non-autoimmune hemolytic anemias. Reduced fluorescence of EMA, typically detected in hereditary spherocytosis, is also seen in CDA II, while reduction of CD55 and CD59 characterizes paroxysmal nocturnal hemoglobinuria (PNH). We studied the flow cytometric profile of EMA, CD55, and CD59 on erythrocytes in CDA III. We found no abnormality of the erythrocyte membrane in CDA III and concluded that standard flow cytometry cannot be used to discriminate between CDA III and normal controls.

In CDA I and CDA II a majority of patients, including those who are not transfusion dependent, suffer from iron overload, which, according to earlier studies, is not the case in CDA III. We found that individuals of the Västerbotten CDA III family carry mutations in the hemochromatosis (HFE) gene. Three CDA III patients with heterozygous or compound HFE mutations need treatment with phlebotomy due to iron overload. One of them carries heterozygous H63D mutation, which is not reported to lead to iron overload by itself in otherwise healthy individuals. We propose that molecular genetic testing of the HFE gene is indicated in all patients with CDA, including CDA III.

Ort, förlag, år, upplaga, sidor
Umeå: print och media, 2016. s. 54
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1784
Nyckelord
congenital dyserythropoietic anemia, KIF23, hereditary hemochromatosis, iron overload, flow cytometry
Nationell ämneskategori
Hematologi
Identifikatorer
urn:nbn:se:umu:diva-117454 (URN)978-91-7601-424-0 (ISBN)
Disputation
2016-04-22, E04, By 6E, 901 85, Norrlands Universitets sjukhus, Umeå, 19:35 (Svenska)
Opponent
Handledare
Tillgänglig från: 2016-04-15 Skapad: 2016-02-29 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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Liljeholm, MariaGrönlund, ElisabethGolovleva, IrinaSandström, HerbertWahlin, Anders

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