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Chapter 19 : Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry

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Abstract:

This chapter focuses on four less common clinical applications of flow cytometry: paroxysmal nocturnal hemoglobinuria (PNH), T-cell receptor Vβ (TCR Vβ) analysis, fetal hemoglobin (Hgb F) detection, and platelet surface marker and functional testing. Three PNH red blood cell (RBC) phenotypes are recognized due to variations in genetic defects that can result in PNH. These are types I, II, and III, which exhibit normal, moderate, and severe complement sensitivities, respectively. Classical clinical tests for PNH are aimed at demonstrating the presence of RBCs that are exceptionally sensitive to the hemolytic action of complement compared to normal RBCs. Flow cytometry is becoming the preferred method for assessing blood samples for the presence of PNH clones. In recent years, monoclonal antibody reagents specific to various TCR-β family V regions have become available. This method offers greater sensitivity and specificity than classical tests in identifying patients with PNH. Structural and functional defects in platelets can lead to a variety of bleeding disorders. Development of commercially available antibodies to relevant platelet antigens permits the discrimination between activated and resting platelets and permits platelet receptor quantitation. Clinical diagnosis is supported by monitoring uptake of mepacrine (a fluorescent molecule that is rapidly taken up and localized to dense granules) into dense granules of platelets and its loss with dense granules release upon stimulation. Idiopathic thrombocytopenic purpura is primarily a disease of increased peripheral platelet destruction, with most patients carrying antibodies to specific platelet membrane glycoproteins, resulting in splenic sequestration and phagocytosis by mononuclear macrophages.

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19

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Figures

Image of FIGURE 1
FIGURE 1

PNH analysis of RBCs. (A) Light scatter gating of RBCs; (B) isotype control; (C) dual-color CD55 and CD59 staining identifies CD55 CD59 RBCs in a PNH patient.

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
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Image of FIGURE 2
FIGURE 2

PNH analysis of neutrophils. (A) CD45 and light scatter gating of neutrophils; (B) iso-type control; (C) two-color CD55 and CD59 staining identifies CD55 CD59 neutrophils in PNH patient.

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
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Image of FIGURE 3
FIGURE 3

Vβ analysis, T large granular lymphocyte leukemia. (A) CD3 gating for analysis. More specific CD4 or CD8 gating may also be done. (B) Dot plot analysis of T-cell gate with Vβ 12 FITC, Vβ 5.2 PE, and Vβ 2 PE/FITC. (C) Bar graph of Vβ expression showing marked overexpression of Vβ 2.

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
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Image of FIGURE 4
FIGURE 4

Hgb F testing. (A) Forward and side scatter gating of RBCs. (B) Cells with high FL2 are excluded from acquisition to avoid problems with autofluorescence. (C) Case with no detectable fetal RBCs. (D) Case with 1.6% fetal RBCs.

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
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Image of FIGURE 5
FIGURE 5

Platelet flow cytometry surface analysis. (A) Light scatter gating of platelets; (B) loss of expression of CD41; and (C) marked decrease in CD61 expression. These features are consistent with Glanzmann’s thrombasthenia.

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
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Image of FIGURE 6
FIGURE 6

Mepacrine release as a measure of granule function. (A) Platelets loaded with mepacrine show normal fluorescence uptake (upper panel) which decreases upon thrombin activation and granule release (lower panel). (B) Abnormal (decreased) mepacrine release compared to normal.

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
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Tables

Generic image for table
TABLE 1

CD markers for GPI-anchored proteins

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
Generic image for table
TABLE 2

Parameters used for gating specific cell types and GPI-linked proteins for PNH screening

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
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TABLE 3

Platelet markers

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
Generic image for table

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
Generic image for table

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19
Generic image for table
TABLE 4

Tube setup for platelet staining protocol

Citation: Kung J, Hsi E. 2006. Nontraditional Clinical Applications: Paroxysmal Nocturnal Hemoglobinuria Testing, Fetal Red Cell Testing, T-Cell Receptor Vβ Analysis, and Platelet Analysis by Flow Cytometry, p 158-170. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch19

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