Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria

Abstract

Within the contemporary multitude of complex methods used in clinical flow cytometry, very few techniques exist which can be described as disease-specific diagnostic tests. Detection of glycophosphatidylinositol (GPI)-linked antigens on hematopoietic cells using monoclonal antibodies and flow cytometry forms the basis of a specific diagnostic test for paroxysmal nocturnal hemoglobinuria (PNH). Absent or markedly diminished expression of GPI-linked antigens is, in the appropriate clinical setting, specific for all patients with PNH. Clinically, PNH is a syndrome characterized by bone marrow failure, acquired hemolytic anemia, and a thrombotic tendency. The molecular genetic lesion responsible for this condition is a somatic mutation of the X-linked pig-a gene within a multipotent hematopoietic stem cell. Due to its rarity, delay in diagnosis is not uncommon for patients with PNH. Once a definitive diagnosis is established, this can make a considerable impact on patient management and prognosis. In this article, we review the complimentary roles that molecular biology and flow cytometry have played in unraveling the genotypic and phenotypic aspects of this unique condition. Cytometry (Comm. Clin. Cytometry) 42:223-233, 2000.

Detailed immunophenotypic characterization of different major and minor subsets of peripheral blood cells in patients with paroxysmal nocturnal hemoglobinuria

“…Our results show that in PNH, CD59 has a predominant unimodal pattern of expression on PLTs, monocytes, CD16 + , and BDCA3 ‐ myeloid DCs, as well as on all lymphoid cell subsets analyzed, in the absence of a clear discrimination between the patients’ PNH and normal residual cells for all PB‐cell subsets, except RBCs and neutrophils. Because of this, routine use of CD59 for the diagnosis of PNH on PB‐cell subsets other than RBCs and neutrophils could be suboptimal, in line with previous aberrations made by others 23,25 . Similarly, expression of CD55 was decreased on RBCs with a unimodal pattern being found in most cases; in addition, CD55 only showed a bimodal pattern of expression among the other PB‐cell populations analyzed in between 50 and 86 percent of the cases, all other PNH patients showing a decreased unimodal pattern of expression of CD55.…”

“…At present, flow cytometric analysis of the expression of GPI‐AP on PB cells is the preferred method for the diagnostic screening of PNH 1,13,17,21,22 . In this regard, both CD55 and CD59 have been considered as the most informative markers 10,14,17,21‐24 . This is due to the fact that both proteins are ubiquitously distributed throughout the different subsets of PB cells 10,11,22 and because their deficient expression on RBCs leads to complement‐mediated intravascular hemolysis, the most frequent clinical manifestation of the disease 10,16 .…”

Paroxysmal Nocturnal Hemoglobinuria is rare cause for thrombosis of the intra‐abdominal veins in the ethnic Indian population – results fromFLAER‐based flowcytometry screening

“…Both of these patients fulfilled the diagnostic criteria of PNH, that is, deficiency of at least two different GPI‐AP, on two different lineages . Details of positive and negative cases are summarized in Tables and , respectively.…”

Recent developments in the understanding and management of paroxysmal nocturnal haemoglobinuria

“…The development of flow cytometry has allowed the detection of small PNH clones which would otherwise not be identified. This has allowed their identification in a proportion of patients with aplastic anaemia and other marrow failure disorders and has led to the classification of PNH into two broad groups: (i) haemolytic PNH, characterised by overt episodes of intravascular haemolysis and typically with large PNH clones and (ii) hypoplastic PNH with a clinical picture dominated by cytopenias, with no overt haemolysis and normally small PNH clones or predominantly Type II cells (Richards et al , 2000). An updated classification system has recently been proposed, which divides patients with PNH clones into the following subgroups (Parker et al , 2005):…”