Laboratory studies for paroxysmal nocturnal hemoglobinuria, with emphasis on flow cytometry

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell disorder caused by somatic mutations in the PIG-A gene, leading to the production of blood cells with absent or decreased expression of glycosylphosphatidylinositol-anchored proteins, including CD55 and CD59. Clinically, PNH is classified into three variants: classic (hemolytic), in the setting of another specified bone marrow disorder (such as aplastic anemia or myelodysplastic syndrome) and subclinical (asymptomatic). PNH testing is recommended for patients with intravascular hemolysis, acquired bone marrow failure syndromes and thrombosis with unusual features. Despite the availability of consensus guidelines for PNH diagnosis and monitoring, there are still discrepancies on how PNH tests are carried out, and these technical variations may lead to an incorrect diagnosis. Herein, we provide a brief historical overview of PNH, focusing on the laboratory tests available and on the current recommendations for PNH diagnosis and monitoring based in flow cytometry.

Keywords: Bone marrow failure syndromes; Flow cytometry; Glycosylphosphatidylinositol; Intravascular hemolysis; PNH; Paroxysmal nocturnal hemoglobinuria.

Fig. 1

Flow cytometry studies for the identification and quantification of GPI-AP deficient RBC. Bivariate dot plots (Panel A) and band diagrams (Panel B) obtained from FCM analysis of the peripheral blood RBC of a healthy individual and of a patient with classic PNH, using the Infinicyt ™ software, version 1.8.0 (Cytognos, Salamanca, Spain). Please note that the normal PB sample has only type I RBC (red dots, normal CD59 expression, 100%), whereas in the PB sample from this PNH patient, type I RBC (red dots, normal CD59 expression, 48.2%) coexist with type II (orange dots, partial CD59 deficiency, 17.4%) and type III (yellow dots, complete CD59 deficiency, 34.4%) RBC. Red blood cells were stained with the following combinations of mAbs: anti-CD59-FITC (clone MEM-43; Invitrogen), anti-CD55-PE (clone IA10; Becton Dickinson), anti-CD235a-APC (clone GA-R2/HIR2; BD Biosciences), and anti-CD45-KO (clone J.33; Immunotech). RBC were washed twice after staining. FCM was performed using a Navios™ flow cytometer (Beckman Coulter). The minimum number of RBC events acquired per tube was of 250,000; whenever possible, a higher number of events was acquired, up to 1,000,000. All the events acquired were recorded and stored as listmode files (.lmd) and then converted into flow cytometry standard (.fcs) 3.0 files for data analysis. FSC and SSC, represented as FSC and SSC integral, were captured on a logarithmic scale; for fluorescence parameters, a logarithmic amplification was also used. RBC were selected based on their light scatter profile, on the absence of CD45 expression, and positivity for CD235a, and then analyzed for the expression of CD55 and CD59. Abbreviations: APC, Allophycocyanin; FCM, Flow Cytometry; FITC, Fluorescein Isothiocyanate; FSC, Forward Scatter; GPI-AP, Glycosylphosphatidylinositol-anchored proteins; KO, Krome orange; PB, peripheral blood; PE, Phycoerythrin; PNH, Paroxysmal nocturnal hemoglobinuria; RBC, Red blood cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Flow cytometry studies for the identification and quantification of GPI-AP deficient WBC cells. Bivariate dot plots (Panel A) and band diagrams (Panel B) obtained by FCM analysis of the peripheral blood WBC of a healthy individual and of a patient with classic PNH, using the Infinicyt ™ software, version 1.8.0 (Cytognos, Salamanca, Spain). Please note that in the normal PB sample neutrophils are FLAER+, CD157+, CD16⁺ and CD66b+ (purple dots) and monocytes are FLAER+, CD157+ and CD14⁺ (red dots). In contrast, in the PB from the PNH patient, normal neutrophils (purple dots; 74.4%) coexist with PNH neutrophils (pink dots; FLAER-, CD157-, CD16⁻, CD66b-; 25.6%) and normal monocytes (red dots; 16.4%) coexist with PNH monocytes (orange dots; FLAER-, CD157-, and CD14⁻; 83.6%). In addition, normal eosinophils (dark blue dots; 70.3%) and PNH eosinophils (light blue dots; 29.7%) also coexist in the patient’ PB sample, whereas lymphocytes (gray dots) are normal in both samples. Cell staining was done using a stain-lyse-and-then-wash method, and the BD FACS™ Lysing Solution (Becton Dickinson), according to the instructions of the manufacturer. Peripheral blood cells were stained with FLAER (Alexa 488; Cerdarlane) and the following combination of mAbs: anti-CD64-PE (clone 22; Immunotech), anti-CD66b-PC5.5 (clone G10F5; Biolegend), anti-CD10-PC7 (clone ALB1; Immunotech), anti-CD157-APC (clone SY11B5; eBiosciences), anti-CD14-APC-H7 (clone MOP9; BD Pharmingen), anti-CD16-V450 (clone 3G8; BD Horizon), and anti-CD45-KO (clone J.33; Immunotech). FCM was performed using a Navios™ flow cytometer (Beckman Coulter). The minimum number of neutrophils and monocyte events acquired per tube was of 250,000; whenever possible, higher numbers of events were acquired, up to 500,000 events for each cell population. All the events acquired were recorded and stored as listmode files (.lmd) and then converted into flow cytometry standard (.fcs) 3.0 files for data analysis. FSC and SSC, represented as FSC integral and SSC peak, were captured on a linear scale; for fluorescence parameters, a logarithmic amplification was used. The WBC populations were selected based on their light scatter profile and on the expression of CD45 and non-GPI-AP (e.g. CD10 and CD64, for neutrophils and monocytes, respectively), and then analyzed for the expression of the GPI-AP mentioned above. Abbreviations: APC, Allophycocyanin; FCM, Flow Cytometry; FITC, Fluorescein Isothiocyanate; FSC, Forward Scatter; GPI-AP, Glycosylphosphatidylinositol-anchored proteins; KO, Krome orange; PB, peripheral blood; PE, Phycoerythrin; PC5.5, PE-Cyanine 5.5; PC7, PE-Cyanine 7; PNH, Paroxysmal nocturnal hemoglobinuria; WBC, White blood cells; V450, Violet 450. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)