Comparison of High Sensitivity and Conventional Flow Cytometry for Diagnosing Overt Paroxysmal Nocturnal Hemoglobinuria and Detecting Minor Paroxysmal Nocturnal Hemoglobinuria Clones
2019; 39(2): 150-157
Ann Lab Med 2019; 39(5): 438-446
Published online September 1, 2019 https://doi.org/10.3343/alm.2019.39.5.438
Copyright © Korean Society for Laboratory Medicine.
Joonhong Park , M.D.1,2,* , Myungshin Kim , M.D.1,2,* , Yonggoo Kim , M.D., Ph.D.1,2 , Kyungja Han , M.D.1,2 , Nack-Gyun Chung , M.D.3 , Bin Cho , M.D.3 , Sung-Eun Lee , M.D.4 , and Jong Wook Lee , M.D., Ph.D.4
1Department of Laboratory Medicine, 2Catholic Genetic Laboratory Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea; 3Department of Pediatrics, College of Medicine, The Catholic University of Korea, Seoul, Korea; 4Division of Hematology, Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
Correspondence to: Yonggoo Kim, M.D., Ph.D.
Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea
Tel: +82-2-2258-1642, Fax: +82-2-2258-1719, E-mail: email@example.com
Jong Wook Lee, M.D., Ph.D.
Division of Hematology, Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea
Tel: +82-2-2258-6050, Fax: +82-2-780-1283, E-mail: firstname.lastname@example.org
*These authors contributed equally to this study.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired pluripotent hematopoietic stem cell disorder associated with an increase in the number of glycosyl-phosphatidyl inositol (GPI)-deficient blood cells. We investigated PNH clonal proliferation in the three cell lineages?granulocytes, T lymphocytes, and red blood cells (RBCs)?by analyzing
Flow cytometry was used on peripheral blood samples from 24 PNH patients to measure the GPI-anchored protein (GPI-AP) deficient fraction in each blood cell lineage.
The GPI-AP deficient fraction among the three lineages was the highest in granulocytes, followed by RBCs and T lymphocytes.
Keywords: Paroxysmal nocturnal hemoglobinuria, PIGA mutation, T-cell receptor clonality
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired pluripotent hematopoietic stem cell (HSC) disorder associated with partial or absolute glycosyl-phosphatidyl inositol-anchored protein (GPI-AP) deficiency [1,2]. In PNH, a somatic defect in HSCs, which synthesize glycosyl-phosphatidyl inositol (GPI), results in defective GPI linkage of decay-accelerating factor (DAF) and membrane inhibitor of reactive lysis (MIRL) in red blood cells (RBCs). The lack of cell surface expression of these proteins leads to cell lysis by the complement system and destruction of large numbers of RBCs, resulting in hemoglobinuria [3,4,5]. The most common cause of PNH is somatic mutations associated with the X-chromosomal gene
The clinical manifestation of PNH involves clonal expansion of GPI-AP blood cells, which is controlled by intrinsic and extrinsic factors that affect cell growth and survival . However, little is known about the clonal expansion of GPI-AP deficient HSCs in relation to
Peripheral blood samples were collected for routine flow cytometry screening for PNH from 24 patients at Seoul St. Mary's Hospital, Seoul, Korea, between January 2010 and April 2012. Samples remaining after the screening test were aliquoted at 200 µL/tube. Two or three microtubes were stored immediately at −20℃ until molecular analysis. Diagnosis was confirmed and assigned according to the guidelines for the diagnosis and management of PNH . At sampling, no patient was actively infected, and no evidence for hereditary bone marrow (BM) failure syndromes was found. All patients provided written informed consent for clinical and molecular analyses. The study protocol was approved by the Institutional Review Board of The Catholic University of Korea, Seoul, Korea (KC12RISE0422).
The patients were classified into two groups based on the presence of cytopenia at diagnosis of PNH: PNH with concomitant AA (PNH/AA, N=12) and classic PNH (N=12). Patients who met at least two of the three peripheral blood cytopenias (Hb level: ≤100 g/L, absolute neutrophil count [ANC]: 0.5–1.5×109/L, and platelet count [PLT]: 20–100×109/L) were classified as PNH/AA . Patients with clinical evidence of intravascular hemolysis without any evidence of other BM failure were classified as classic PNH. There were significant differences between the groups in ANC and PLT. The number of patients who underwent previous immunosuppressive therapy and/or corticosteroid treatment was significantly higher in the PNH/AA than in the classic PNH group. The percentage of patients who were treated with eculizumab was higher in the classic PNH group, whereas allogeneic hematopoietic stem cell transplantation (HSCT) was performed exclusively in the PNA/AA group (Table 1).
GPI-AP deficient granulocytes, T lymphocytes, and RBCs were analyzed by flow cytometry using specific monoclonal antibody cocktails: fluorescent aerolysin reagent (FLAER)-Ax488/CD24-PE for granulocytes, FLAER-Ax488/CD3-PE for T lymphocytes, and CD55-FITC/CD59-PE for RBCs. The FLAER-based flow-cytometric assay is recommended to screen for GPI-AP deficient white blood cells (WBCs) because it has higher sensitivity than that based on other GPI-APs, including CD55, CD59, CD24, and CD14, and can detect small clone sizes in PNH as well as AA or myelodysplastic syndrome [11,12].
To analyze GPI-AP deficient granulocytes and T lymphocytes, 5 µL of appropriate monoclonal antibody was added to 0.5×106 WBCs and incubated for 20 minutes at 24℃. After RBCs were lysed using 2 mL of BD fluorescence activated cell sorter (FACS) Lysing Solution (BD Pharmingen, Franklin Lakes, NJ, USA), the cells were washed and analyzed on a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA), according to the manufacturer's recommendation. For each tube, a minimum of 50,000 events for granulocytes or T lymphocytes was routinely collected. To analyze GPI-AP deficient RBCs, 20 µL of whole blood was diluted with 3 mL of phosphate-buffered saline (PBS) followed by the addition of 50 µL to one tube, and 5 µL of CD59-FITC and CD55-PE to the second tube. The samples were incubated in the dark for 60 minutes at 24℃ and washed twice with PBS by centrifugation as required to optimize separation of Type I, II, and III GPI-AP deficient RBCs. After washing, the RBC pellet was re-suspended in 0.2 mL of PBS, and 50,000 RBCs were acquired in the list mode.
Granulocytes and lymphocytes were initially selected by forward scatter and side scatter. Each cell lineage was assessed for combined expression of FLAER/CD24 (granulocytes) and FLAER/CD3 (T lymphocytes). The limit of detection (LOD) using FLAER as the most discriminant marker combined with CD24 or CD3 was determined as 0.1%. However, because FLAER affinity seems restricted to certain types of GPI anchors, making it unsuitable for evaluation of RBCs , we used both CD55 and CD59 for expression analysis for GPI-AP-deficient RBCs with an LOD of 3%.
Granulocytes were isolated from EDTA-blood samples via standard density gradient separation using a mixture of sodium metrizoate and dextran 500 . T lymphocytes were isolated from EDTA-blood samples using the EasySep Human T Cell Enrichment Kit (STEMCELL Technologies, Vancouver, Canada), according to the manufacturer's protocol. GPI-AP deficient granulocytes and T lymphocytes were additionally sorted into the FLAER(−)/CD24 and FLAER(−)/CD24 CD3(+) cell population, respectively, by flow cytometry using BD FACSAria II (BD Biosciences). Genomic DNA was extracted from the sorted GPI-AP deficient granulocytes and T lymphocytes using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany).
Genomic DNA isolated from granulocytes and T lymphocytes was used as a template for amplifying individual exons of
The TCR gene rearrangement assay was used to evaluate the clonality of GPI-AP deficient T lymphocytes. PCR was conducted on genomic DNA isolated from sorted GPI-AP deficient T lymphocytes using IdentiClone
As the laboratory findings were not normally distributed as indicated by Kolmogorov-Smirnov and Shapiro-Wilk tests, continuous variables were summarized as median with range and were compared using the Mann–Whitney U test. Pearson's chi-square test was used to compare categorical variables. Spearman's correlation was determined to estimate the strength and direction of an association that existed between two continuous variables, and a line of best fit was drawn through the data of two variables in a scatterplot. All tests were two-tailed, and
We excluded the patients treated with eculizumab from PNH clone size analysis according to cell lineage because eculizumab promotes the survival of GPI-AP-deficient RBCs compared with that of WBCs or lymphocytes . The GPI-AP deficient cell fraction varied according to patient and cell types. The median GPI-AP deficient cell fraction was the highest in granulocytes followed by RBCs and T lymphocytes. GPI-AP-deficient RBCs were more numerous in classic PNH than in PNH/AA patients (
Nineteen (79.2%) of the 24 PNH patients carried
The incidence of
TCR clonality was detected in 19 (79.2%) out of the 24 PNH patients.
Individual somatic mutations throughout the coding region of the
The mechanisms of clonal expansion have been studied actively to elucidate the pathogenesis of PNH [6,7,22]. The consensus is that a
Consistent with previous studies, we revealed that
Although both the GPI-AP deficient fraction and the
Potential limitations of this study include no identification of
In conclusion, we revealed the presence of
Spearman's correlations between GPI-AP deficient fractions of each cell lineage. (A) Correlation between GPI-AP-deficient fraction of RBCs (%) and GPI-AP-deficient fraction of granulocytes (%). (B) Correlation between GPI-AP deficient fraction of RBCs (%) and reticulocytes (%).
Abbreviations: GPI-AP, glycosyl-phosphatidyl inositol-anchored protein; RBCs, red blood cells.
Spearman's correlations between
Abbreviations: GPI-AP, glycosyl-phosphatidyl inositol-anchored protein; RBCs, red blood cells.
Abbreviation: GPI-AP, glycosyl-phosphatidyl inositol-anchored protein.