Author/Editor		Mlakar, Uroš; Žontar, Darja
Title		Ugotavljanje in spremljanje klona PNH z določanjem CD55 in CD59 na nevtrofilcih
Translated title		Detection and follow up of PNH clone by measuring CD55 and CD59 expression on neutrophils
Type		članek
Source		Zdrav Vestn
Vol. and No.		Letnik 73, št. Suppl 1
Publication year		2004
Volume		str. I-93-6
Language		slo
Abstract		Background. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal haematopoietic stem cell disorder characterised by intravascular haemolysis, bone marrow failure and increased tendency to thrombosis. It is caused by a somatic mutation in the PIG-A gene, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in a clone of blood cells with total or partial deficiency of membrane proteins anchored to the cell surface through GPI anchor. For many years, an increased susceptibility of the PNH red cells to complement lysis in acidified serum (Ham's test) has been essential for diagnosis of the PNH Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of the specific GPI anchor proteins on blood cells. We evaluated a three-colour flow cytometry method for detection and quantification of CD55/59 negative netrophils. Patients and methods. Nine patients (6 with PNH and 3 with aplastic anaemia) who were previously diagnosed as having PNH or aplastic anaemia were evaluated. In the period of three years the patients were serially evaluated for the extent of PNH clone by quantification of CD55/59 negative neutrophils. Three-colour flow cytometry method was used for quantification of the CD55/CD59 negative neutrophils. We used directly conjugated monoclonal antibodies anti-DC15PC5 for identification of neutrophils and anti-CD59-FITC and anti-CD55-PE for the GPI-linked antigens. Results. Patients with haemolytic PNH had > 50% CD59/CD55 negative granulocytes. The proportion of the PNH granulocytes was higher in the patient with frequent and serious haemolytic attacks. Over the period of three years slow growth of the PNH clone was seen in two cases. Two patients with PNH diagnosed 19 years ago and in remission at the time of flow cytometric analysis was devoid of the PNH clone. (Abstract truncated at 2000 characters).
Summary		Izhodišča. Paroksizmalna nočna hemoglobinurija (PNH) je pridobljena klonska bolezen krvotvornih matičnih celic. Zanjo je značilna intravaskularna hemoliza, odpoved kostnega mozga in nagnjenost k trombozam. Nastane zaradi somatske mutacije gena PIGA. Ta je odgovoren za nastanek encima, ki sodeluje pri sintezi glikozilfosfatidilinozitola (GPI). Tako nastane klon krvnih celic, ki na površini nima tistih beljakovin, katerih vezavo omogoča GPI. Vrsto let se je za ugotavljanje PNH uporabljal Hamov preizkus, ki temelji na povečani dovzetnosti eritrocitov za litični učinek komplementa po dodatku kisline. Danes ugotavljamo PNH s pretočno citometrijo, kjer s protitelesi ugotavljamo pomanjkanje beljakovin, odvisnih od GPI. Prikazujemo naše izkušnje z uporabo tribarvne citometrije za ugotavljanje in oceno količine CD55/59 negativnih nevtrofilcev. Bolniki in metode. Obravnavali smo devet bolnikov, ki smo jim že ugotovili PNH ali aplastično anemijo (6 s PNH in 3 z aplastično anemijo). Bolnikom smo v obdobju treh let merili velikost klona PNH z določanjem deleža CD55/59 negativnih nevtrofilcev. Velikost klona smo določali s tribarvno pretočno citometrijo, pri tem smo za ugotavljanje nevtrofilcev uporabili monoklonska protitelesa anti-DC15-PC5, za ugotavljanje beljakovin, odvisnih od GPI, pa anti-CD59-FITC in anti-CD55-PE. Rezultati. Bolniki s hemolitično obliko PNH so imeli več kot 50% CD59/CD55 negativnih nevtrofilcev. Bolnik z največjim klonom je imel najbolj aktivno bolezen s pogostimi hudimi napadi hemolize. V triletnem obdobju smo pri dveh bolnikih opazovali postopno večanje klona PNH. Pri dveh bolnikih z remisijo PNH nismo ugotovili napake PNH. Trije bolniki z aplastično anemijo (hipoplastična PNH) so imeli < 40% CD55/59 negativnih nevtrofilcev. Pri enem bolniku je prišlo do porasta klona PNH. Zaključki. (Izvleček skrajšan pri 2000 znakih).
Descriptors		HEMOGLOBINURIA, PAROXYSMAL ANEMIA, APLASTIC NEUTROPHILS ANTIGENS, CD55 ANTIGENS, CD59 FLOW CYTOMETRY