Multiparametric analysis
Single-tube workflow for neutrophils and monocytes
High sensitivity detection
Reliable identification of
GPI-anchor deficient populations
Optimized workflow
Ready-to-use liquid and
lyophilized formats
Research focused
Designed for advanced flow
cytometry applications
PNH Pathology
Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare disorder that causes the destruction of blood cells: red blood cells (erythrocytes), which are responsible for oxygen transport; white blood cells (leukocytes), cells of the immune system; and platelets (thrombocytes), cell fragments that play a role in blood clotting and tissue repair.
PNH is characterized, as its name suggests, by the presence of hemoglobin in the urine (hemoglobinuria) in an intermittent (paroxysmal) and nighttime (nocturnal) pattern; this hemoglobin comes from destroyed red blood cells (hemolysis).
Related conditions: The early destruction of healthy cells can lead to several disorders:
- Hemolysis, when occurring massively, causes hemolytic anemia due to a reduction in red blood cell levels.
- When cell destruction is widespread and occurs very early, it prevents the generation of new cells in the bone marrow, excessively reducing their number (pancytopenia); this leads to bone marrow aplasia (aplastic anemia).
- The release of hemoglobin causes dysregulation of nitric oxide in the blood, decreasing its levels. Nitric oxide inhibits platelet aggregation and adhesion to each other and to blood vessels; without this inhibition, there is a risk of thrombosis, the formation of clots that block blood flow.
Cause and mechanism of the disease: Cell destruction occurs through activation of the complement system, an innate immune system. This system is based on certain circulating proteins that bind to a pathogen through different activation pathways; this triggers a cascade of multiple reactions involving proteins, convertase enzymes, and protein-derived fragments. The chain reaction culminates in the formation of a Membrane Attack Complex (MAC), composed of several linked fragments that perforate and destroy the target cell.
Complement fragments
Fragments of the complement system’s proteins that bind to the target in order to eliminate it.
GPI-AP
Certain proteins require a GPI anchor to bind to the membrane.
GPI anchor
Glycosylphosphatidylinositol (GPI) is a glycolipid that acts as an anchor between a protein and the cell membrane. It is synthesised by a series of proteins encoded by PIG (phosphatidylinositol glycan) genes.
Transmembrane protein
Some surface proteins are directly attached to the cell membrane via the protein’s own transmembrane domains; they do not rely on other anchoring mechanisms, such as GPI.
MAC
The Membrane Attack Complex is the final structure of the complement system, formed by the sequential assembly of its fragments. The purpose of this complex is to perforate the membrane, thereby causing cell lysis.
Cell lysis
With the formation of the MAC, the lytic function of the complement system is completed, leading to membrane destruction and cell death.
Diagnosis by flow cytometry
The gold standard diagnostic test for PNH is flow cytometry. This test is carried out once other possible causes of anaemia have been ruled out and following a negative result in the direct Coombs test (direct antiglobulin test or DAT), which detects the presence of antibodies on the surface of red blood cells in cases of haemolysis.
PNH clones that is, populations that test positive for the disease can be classified according to their symptoms:
- Classic or haemolytic PNH: intravascular haemolysis without bone marrow involvement.
- PNH with bone marrow involvement: aplastic anaemia or myelodysplastic syndrome (MDS).
- Asymptomatic PNH: no evidence of haemolysis or thrombosis, despite the identification of PNH clones.
Another possible classification relates to the level of GPI expression:
Finally, they can be classified according to the clone size analysed within a target population:
A diagnosis of PNH may refer to a combination of characteristics of the cells analysed, namely whether or not PNH clones are present, the size of the detected clone (the ratio of healthy to diseased cells) and the level of GPI expression in that clone.
The severity of the disease increases with a larger clone size (complete PNH+ or high partial PNH+) and a greater absence of GPI (Type III).
Detection of PNH in red blood cells and analysis strategy
In erythrocytes, a GPI-independent marker is used for comprehensive identification; this is CD235a. CD59 (and/or CD55) is then used to assess the level of GPI expression. GPI deficiency may be complete or partial; therefore, erythrocytes are classified according to GPI-AP expression: Type I, Type II and Type III. (Figure 1, A).
Subsequently, the size of the PNH-positive clones (Type II and Type III) is assessed to determine the extent of the disease, i.e. whether all cells are diseased (complete PNH+) or whether some are healthy (partial PNH+), and what the ratio of diseased to healthy cells is. (Figure 1, B)
Figure 1. A) Identification of CD235a+ erythrocyte subtypes according to their level of CD59 expression. B) Analysis of the size of each cell type.
Detection of PNH in leukocytes and analysis strategy
FLAER
Fluorescein-Labelled Aerolysin is a fluorescently labelled bacterial toxin. This toxin binds specifically to GPI.
CD15
A carbohydrate found in abundance in neutrophils; although it is also present in monocytes and eosinophils, its expression is lower and it can be easily distinguished from neutrophils.
CD64
A transmembrane protein, GPI-independent, specific to the monocyte population.
CD157
GPI-AP which is present in myeloid cells.
CD45
A transmembrane protein present in all white blood cells (pan-leukocyte marker).
A pan-leukocyte marker, CD45, is used to detect leukocytes (Figure 2, A). The populations of interest are then separated using specific markers: CD15 for neutrophils (Figure 2, B) and CD64 for monocytes (Figure 2, C). These three markers are GPI-independent. PNH clones are identified using GPI-AP, CD157 (common to myeloid lineage cells, such as neutrophils and monocytes), and FLAER, a molecule that binds specifically to GPI. When used in combination, the clone size and the level of expression can be assessed.
The FLAER/CD157 analysis can yield several interpretations:
- A healthy cell should express both GPI and CD157, so a double-positive (DP) result will be observed in the FLAER/CD157 analysis.
- A Type III PNH clone does not express GPI and, therefore, will not express CD157. This results in a double-negative (DN) result.
- A Type II PNH clone will partially express GPI. It is common for these clones to be only weakly positive for FLAER and negative for CD157. This is because FLAER has high sensitivity and detects all GPI molecules. As CD157 is present in lower proportions and the antibody used is not as sensitive, the signal is undetectable. In this case, we are dealing with a single positive (SP).
- The clone size can be assessed based on the presence of one or more populations. A single DP population indicates PNH-, a single DN population indicates complete PNH+, and two populations (DP and DN) indicate partial PNH+.
In leukocytes, analysis of the clone size is relevant; the level of expression is more easily analysed in erythrocytes, although it can also be carried out there.
ANALYSIS LEUKOCYTES
Figure 2. Analysis of PNH clones in leukocytes. A) Separation of CD45+ leukocytes into two populations of interest: monocytes (CD64+/CD15-) and neutrophils (CD15+/CD64-). B) and C) Identification of neutrophils (B) and monocytes (C), depending on whether they are healthy cells (FLAER+/CD157+, DP) or pathological cells (FLAER-/CD157-, DN).
