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Chapter 18 : High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry

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High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, Page 1 of 2

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Abstract:

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening acquired hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositol-glycan complementation class A (PIG-A) gene (1–4). In normal individuals, this gene encodes an enzyme involved in the first stage of glycophosphatidylinositol (GPI) biosynthesis. In PNH, as a result of the mutation(s) in the PIG-A gene, there is a partial or absolute inability to make GPI-anchored proteins, including complement-defense structures such as CD55 and CD59 on red blood cells (RBCs) and white blood cells (WBCs) (5–8). Absence of CD59 in particular (9, 10) and CD55 on RBCs is largely responsible for intravascular hemolysis associated with clinical PNH (reviewed in reference 11).

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
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Figures

Image of FIGURE 1
FIGURE 1

(A) Instrument setup for high-sensitivity RBC assay. Light-scatter voltages were established in logarithmic mode such that RBCs from a diluted normal PB sample clustered in the middle of the plot (left). Gated RBCs from region R1 were displayed on FL-1 versus FL-2 plot and PMT voltages were adjusted to get the unstained RBCs properly on-scale (middle). Samples were single stained with either CD235aFITC (top right) or CD59PE (bottom right) and compensation was adjusted to reduce spectral overlap. (B) Analysis of normal RBCs. Normal RBCs stained and acquired using the instrument settings established in panel A. RBCs gated in R1 (left) and displayed on CD235aFITC versus FS plot (middle). CD235a+ RBCs were gated in R2 and aggregated RBCs (see arrow; 0.23% of total) were excluded as shown. Cells from R1 and R2 were displayed on CD235aFITC versus CD59PE (right). A second sample stained only with CD235aFITC was analyzed to approximate the location of the type III gate (gate III). (C) Analysis of fresh PNH sample. A PNH sample was stained as described with the optimized RBC protocol as outlined in the Methods, and analyzed as described for the normal sample (panel B). Aggregated RBCs (0.43%) were excluded by gate R2. The sample contained 42.47% PNH type III cells and 6.02% PNH type II cells.

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
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Image of FIGURE 2
FIGURE 2

Example of high-sensitivity neutrophil assay. A sample from a long-term PNH patient was stained with FLAER, CD24PE, CD15PECy5, and CD45PECy7. Light-scatter voltages were established so that all nucleated cells were visible above the forward-scatter threshold (top left) and debris was excluded with a combination of light scatter and CD45 gating (top middle). CD45+ events were displayed on CD15 versus SS plot (top right) and neutrophils (bright CD15, high SS) were gated. Neutrophils displayed on a FLAER versus CD24 plot (bottom row, right) and PNH neutrophils (FLAER-negative, CD24-negative) were enumerated in the lower left quadrant. Normal neutrophils were enumerated in the upper right quadrant. Lymphocytes were gated by a combination of CD45/SS staining (top middle plot) and lack of CD15 expression (not shown) and assessed for PNH phenotypes in the lower-left quadrant. Normal T lymphocytes (FLAER+, CD24-negative) are visible in the lower right quadrant and normal B lymphocytes (FLAER+, CD24+) are visible on the upper right quadrant.

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
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Image of FIGURE 3
FIGURE 3

Example of high-sensitivity monocyte assay. A sample from a long-term PNH patient was stained with FLAER, CD14PE, CD64PECy5, and CD45PECy7 and analyzed in a manner similar to that shown for the neutrophil assay in Fig. 2 . CD45+ events were displayed on CD64 versus SS plot (top right) and monocytes (bright CD64, intermediate SS) were gated. Monocytes were displayed on a FLAER versus CD14 plot (bottom row, right) and the PNH monocytes (FLAER-negative, CD14-negative) were enumerated in the lower left quadrant. Lymphocytes were gated by a combination of CD45/SS staining (top-middle plot) and lack of CD64 expression (not shown) and assessed for PNH phenotypes in the lower left quadrant. Normal lymphocytes (FLAER+, CD14-negative) are visible in the lower right quadrant.

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
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Image of FIGURE 4
FIGURE 4

(A) Example of normal sample stained with high-sensitivity neutrophil assay. Normal sample stained and analyzed exactly as described in Fig. 2 . Gated neutrophils displayed on FLAER versus CD24 plot (right) and zero PNH neutrophils were detected in the lower left quadrant. (B) Example of normal sample stained with the high-sensitivity monocyte assay. Normal sample stained and analyzed exactly as described in Fig. 3 . Gated monocytes displayed on FLAER versus CD14 plot (right) and only 1 PNH monocyte phenotype was detected in the lower left quadrant.

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
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Image of FIGURE 5
FIGURE 5

Five-color, single-tube, CD157-based assay for PNH neutrophil and monocytes. A fresh sample from a long-term PNH patient was stained with FLAER, CD157PE, CD64ECD, CD15PECy5, and CD45PECy7 and data were acquired on FC500. Debris was removed with light scatter (top row, left) and CD45 gating (top row, middle). Neutrophils (middle row, left) and monocytes (middle row, middle) were identified and gated based on CD15 and CD64 expression, respectively. Gated neutrophils (bottom row, left) and monocytes (bottom row, middle) were displayed on FLAER versus CD157 plots and PNH neutrophils and monocytes (FLAER-negative, CD157-negative) were identified. Gated lymphocytes (internal controls) were identified and sequentially gated based on CD45 and SS (top row middle) and lack of CD64 staining (not shown) and displayed on FLAER versus CD157 (top row, right) or FLAER versus CD64 (middle row, right) and FLAER versus CD15 (bottom row, right).

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
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Image of FIGURE 6
FIGURE 6

Six-color single-tube assay for PNH neutrophil and monocytes. Fresh PNH sample stained with FLAER, CD24PE, CD15PECy5, CD64PC7, CD14APC-A700, and CD45KO. Data acquired on Navios cytometer equipped with three lasers and 10 PMTs. Neutrophils were gated by sequential analysis of light scatter, CD45 gating, and CD15 expression (top row). Gated neutrophils were displayed on FLAER versus CD24PE plot (top right) to delineate PNH neutrophils (lower left quadrant) from normal neutrophils (upper right quadrant). Monocytes were sequentially gated by light scatter, CD45, and bright CD64 expression (bottom row, left). CD64-gated monocytes were displayed on adjacent FLAER versus CD14APC-A700 plot and PNH monocytes (lower left quadrant) were delineated from normal monocytes (upper right quadrant). Lymphocytes were gated by a combination of light scatter and CD45/SS staining and displayed on four bivariate dot plots (right lower and right bottom rows) displaying FLAER versus CD24, CD14, CD15, and CD64 to confirm optimal instrument setup/compensation and validate the performance of all antibody conjugate used in the assay.

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
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Image of FIGURE 7
FIGURE 7

Examples of two samples containing type II neutrophils and monocytes. Samples stained with five-color CD157-based assay (top row) or four-color neutrophil (bottom left plot) or four-color monocyte (bottom right plot) assays. CD157 separates PNH type II neutrophils from type III cells more effectively than CD24. When major populations of type II cells are present, as shown in case 1, this can lead to difficulty in delineating type I cells from PNH type II cells. This issue is also apparent in comparing CD157 (top right) and CD14 staining (bottom right) of monocytes.

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
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References

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Tables

Generic image for table
TABLE 1

Recommended antibody conjugates for high-sensitivity detection of PNH RBCs

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
Generic image for table
TABLE 2

Recommended reagent conjugates for high-sensitivity detection of PNH WBCs: Beckman Coulter instruments

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18
Generic image for table
TABLE 3

Recommended reagent conjugates for high-sensitivity detection of PNH WBCs: Becton Dickinson instruments

Citation: Illingworth A, Keeney M, Sutherland D. 2016. High-Sensitivity Detection of Red and White Blood Cells in Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry, p 168-181. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch18

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