Bessler and Hiken (pp 104–110) Figure 3.
Bessler and Hiken (pp 104–110) Figure 3. Diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). (A) Ham test, the traditional clinical assay for diagnosis of PNH, is based on the increased susceptibility of PNH red blood cells to complement-mediated lysis. PNH (tubes 1, 2) and control (tubes 3, 4) red blood cells are incubated with serum (S), or serum in which complement has been heat inactivated (HS). Cells and debris are pelleted, and the degree of lysis is assessed by the presence of red pigment from hemoglobin in the serum. Only PNH red blood cells show complement-dependent lysis (tube 1), due to their lack of expression of GPI-linked complement inhibitors CD55 and CD59, while control red blood cells are protected (tube 3). (B) Granulocytes from a PNH patient were stained with fluorescently labeled antibody against the GPI-linked protein CD16. Strongly positive (normal) and completely negative (PNH) cells are present. Typically, populations of both normal and PNH hematopoietic cells reside simultaneously in the peripheral blood. (C) Diagnosis of PNH by using flow cytometry. All hematopoietic lineages can be affected by PNH. Currently, clinical assays often assess PNH based on staining of white blood cells with fluorescently labeled antibodies against at least two GPI-linked proteins or, as shown here using FLAER, a fluorescently labeled derivative of the bacterial protein aerolysin, which naturally binds the GPI anchor. On the left the dot plot analysis of total peripheral blood white blood cells (WBC) from PNH (top) and control (bottom) individuals are shown. Note that in the sample from the PNH patient a proportion of cells have a decreased binding of FLAER (PNH cells). To the right the histograms of specific white blood cell lineages seen in the associated dot plots show substantially reduced FLAER binding to PNH (top) on 95% of neutrophils (PMN) and monocytes (Mono), 15% of lymphocytes (Lymph), as compared to control (bottom). (D) FACS analysis of red blood cells using fluorescently labeled anti-CD59 is used to assess the PNH red cell population and the degree of GPI-anchor deficiency. Normal individuals show uniformly strong staining of red blood cells with CD59 ((i), hatched line shows isotype-matched control antibody). A classic PNH patient (ii) shows distinct populations of type III cells, which are completely deficient in surface expression of GPI-linked proteins, and type I cells, which show normal expression of GPI-linked proteins. Type II cells show a partial deficiency of GPI-linked protein surface expression and can be seen in PNH patients either alone (iii) or in combination with type III and/or type I populations (iv).

Diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). (A) Ham test, the traditional clinical assay for diagnosis of PNH, is based on the increased susceptibility of PNH red blood cells to complement-mediated lysis. PNH (tubes 1, 2) and control (tubes 3, 4) red blood cells are incubated with serum (S), or serum in which complement has been heat inactivated (HS). Cells and debris are pelleted, and the degree of lysis is assessed by the presence of red pigment from hemoglobin in the serum. Only PNH red blood cells show complement-dependent lysis (tube 1), due to their lack of expression of GPI-linked complement inhibitors CD55 and CD59, while control red blood cells are protected (tube 3). (B) Granulocytes from a PNH patient were stained with fluorescently labeled antibody against the GPI-linked protein CD16. Strongly positive (normal) and completely negative (PNH) cells are present. Typically, populations of both normal and PNH hematopoietic cells reside simultaneously in the peripheral blood. (C) Diagnosis of PNH by using flow cytometry. All hematopoietic lineages can be affected by PNH. Currently, clinical assays often assess PNH based on staining of white blood cells with fluorescently labeled antibodies against at least two GPI-linked proteins or, as shown here using FLAER, a fluorescently labeled derivative of the bacterial protein aerolysin, which naturally binds the GPI anchor. On the left the dot plot analysis of total peripheral blood white blood cells (WBC) from PNH (top) and control (bottom) individuals are shown. Note that in the sample from the PNH patient a proportion of cells have a decreased binding of FLAER (PNH cells). To the right the histograms of specific white blood cell lineages seen in the associated dot plots show substantially reduced FLAER binding to PNH (top) on 95% of neutrophils (PMN) and monocytes (Mono), 15% of lymphocytes (Lymph), as compared to control (bottom). (D) FACS analysis of red blood cells using fluorescently labeled anti-CD59 is used to assess the PNH red cell population and the degree of GPI-anchor deficiency. Normal individuals show uniformly strong staining of red blood cells with CD59 ((i), hatched line shows isotype-matched control antibody). A classic PNH patient (ii) shows distinct populations of type III cells, which are completely deficient in surface expression of GPI-linked proteins, and type I cells, which show normal expression of GPI-linked proteins. Type II cells show a partial deficiency of GPI-linked protein surface expression and can be seen in PNH patients either alone (iii) or in combination with type III and/or type I populations (iv).

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