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. Author manuscript; available in PMC: 2014 Aug 7.
Published in final edited form as: Am J Clin Pathol. 2000 Sep;114(3):459–466. doi: 10.1093/ajcp/114.3.459

Improved Detection and Characterization of Paroxysmal Nocturnal Hemoglobinuria Using Fluorescent Aerolysin

Robert A Brodsky 1,*, Galina L Mukhina 1, Shiyong Li 2, Kim L Nelson 3, Patricia L Chiurazzi 2, J Thomas Buckley 3, Michael J Borowitz 2
PMCID: PMC4124633  NIHMSID: NIHMS401601  PMID: 10989647

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.

Keywords: Paroxysmal nocturnal hemoglobinuria, Aerolysin, GPI anchor proteins, CD59, Aplastic anemia

Paroxysmal nocturnal hemoglobinuria (PNH) is a unique clonal hematopoietic stem cell disorder distinguished by a relative or absolute deficiency of all glycosylphosphatidylinositol (GPI) anchor membrane proteins.1–3 The cause of the deficiency is a somatic mutation in the X-linked gene PIGA, which encodes the enzyme required for the first step in the biosynthesis of GPI anchors.4–8 GPI anchors attach more than a dozen proteins to the surfaces of hematopoietic cells, including the complement regulatory proteins CD55 and CD59.1 These proteins are not displayed on PNH blood cells as a consequence of the PIGA mutation.

The use of labeled monoclonal antibodies to detect deficiencies of individual GPI anchor proteins on circulating cells by flow cytometry has become the standard means to diagnose PNH.9–11 This technology can distinguish among 3 types of cells: cells with nearly normal expression of GPI anchor proteins (type I cells), cells with intermediate expression of GPI anchor proteins (type II cells), and cells with no expression of GPI anchor proteins (type III cells). However, there is no single monoclonal antibody-based flow cytometric test that can be used to establish the diagnosis unequivocally, since different hematopoietic cell lineages display different arrays of GPI anchor proteins, and certain proteins, such as CD5812 and CD16,13 can be expressed on the cell surface in both a GPI anchor and a transmembrane form. Thus, it has been recommended that at least 2 different monoclonal antibodies directed against 2 different GPI anchor proteins on at least 2 different cell lineages must be used for a definitive diagnosis of PNH.9–11

Unlike monoclonal antibodies, which each bind to a single GPI anchor protein, the channel-forming toxin, aerolysin, and its inactive precursor, proaerolysin, have the remarkable ability to bind selectively and with high affinity to the GPI anchor itself.14–16 As a result, they can be used to detect a wide variety of GPI anchor proteins. It seems likely that the protein binds to the core of the anchor (ethanolamine-HPO4-6Man-alpha-1-2Man-alpha-1-6Man-alpha-1-4G1cNH21-6-myo-inositol 1HPO4), which is conserved from species to species and from cell to cell. Because PNH cells lack GPI anchor proteins, aerolysin and proaerolysin are unable to bind to them, and, as a result, these cells are far less sensitive to the toxin than normal cells. Brodsky et al16 recently demonstrated that the resistance of PNH cells to aerolysin provides a basis for a simple biologic assay using erythrocytes that is sensitive and specific for the diagnosis of PNH.16

The PNH defect is most accurately measured by analyzing nucleated cells, such as granulocytes and monocytes, rather than erythrocytes. Erythrocytes are unreliable indicators because patients with PNH frequently receive erythrocyte transfusions and also because type III PNH erythrocytes have a shorter life-span than type I cells owing to their increased sensitivity to complement. Herein we describe a fluorescently labeled variant of proaerolysin that can accurately detect GPI protein deficiency in a variety of cell types, thus forming the basis for an improved diagnostic test for PNH and providing a new tool to study GPI anchor proteins.

Materials and Methods

Production of Fluorescent Proaerolysin

Nucleated cells are able to convert proaerolysin to aerolysin by using furin or other proteases,17 so that they are sensitive to both forms of the protein. In these studies, we used a proaerolysin variant (T253/A300C) that has the same affinity for GPI anchor proteins as wild-type proaerolysin18 but that is completely inactive even after it is converted to the aerolysin form.19 The double mutation, created by site-directed mutagenesis, blocks a critical step in channel formation.19 The purified variant protein was labeled with the green chromophore Alexa Fluor 488 (Molecular Probes, Eugene, OR) following the instructions supplied by the manufacturer. The resulting fluorescent proaerolysin variant (FLAER), which has fluorescent properties comparable to fluorescein isothiocyanate (FITC) proteins, is available from Protox Biotech (Victoria, British Columbia; web.uvic.ca/idc/protox/-protox.html).

Cell Lines

The GPI anchor–deficient B-lymphoblastoid cell line, LD−, harbors a previously characterized PIGA mutation.8 LD−(PIGA+) is a GPI anchor–replete cell line that was established by stable transfection of an expression vector containing the full-length PIGA complementary DNA (cDNA) into the LD− cell line.20 Both cell lines were maintained in RPMI 1640 medium (GIBCO, Gaithersburg, MD) with 10% heat-inactivated fetal calf serum. To measure CD59 expression, cells were washed in cold RPMI with 0.2% fetal calf serum, stained with an FITC-conjugated monoclonal antibody to CD59 (Research Diagnostics, Flanders, NJ) at 4°C, and analyzed by flow cytometry (FACscan, Becton Dickinson, San Jose, CA). To measure GPI anchor expression, cells were washed with RPMI and incubated with FLAER for 40 minutes at room temperature. Cells were washed twice with cold phosphate-buffered saline (PBS) and fluorescence intensity was measured by flow cytometry. The murine cell lines EL4 and EL4 (Thy-1-f) were generously supplied by Robert Hyman, PhD (Salk Institute, La Jolla, CA). The EL4 cell line expresses GPI-anchored proteins; the EL4 (Thy-1-f) is GPI anchor deficient. Both murine cell lines were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% bovine fetal clone I serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin in 5% carbon dioxide at 37°C.

Confocal Fluorescence Microscopy

EL4 and EL4 (Thy-l-f) cells were suspended at 106 cells per milliliter in DMEM 0.5% bovine serum albumin and labeled with 10−8 mol/L FLAER for 1 hour at 4°C. Cells were then washed twice in PBS and fixed in 4% paraformaldehyde in PBS for 30 minutes at room temperature. Cells were washed twice in PBS and visualized with a laser scanning confocal microscope after a 1:1 dilution with 2.3% wt/vol DABCO (1,4-diazobicyclo [2,2,2] octane), in a 20-mmol/L concentration of tris(hydroxymethyl)aminomethane, pH 8, and 90% glycerol.

Patients

Venous peripheral blood from patients and healthy control subjects was drawn into EDTA-containing tubes after informed consent as approved by the Joint Committee on Clinical Investigation of the Johns Hopkins Hospital, Baltimore, MD. A previous diagnosis of PNH was made in 8 patients at Johns Hopkins Hospital; 3 patients had a previous diagnosis of aplastic anemia, and 3 were diagnosed previously with a myelodysplastic syndrome.

Phenotyping of Primary PNH Cells

Binding of mutant aerolysin to leukocyte subpopulations was performed by 3-color flow cytometry using anti-CD45-perCP and FLAER or FITC-conjugated anti-CD59 combined with anti-CD 15, anti-CD33, anti-CD3, or anti-CD19 conjugated to phycoerythrin. In some experiments, 4-color flow cytometry was used, combining FLAER or anti-CD59-FITC, anti-CD33-PE, anti-CD45-perCP, and anti-CD14-APC, to compare expression of either CD59 or FLAER with that of the GPI anchored monocyte marker CD14. Briefly, RBCs from aliquots of peripheral blood were lysed with ammonium chloride. After washing FLAER was added to the resulting cell pellet and the mixture incubated in the dark at room temperature. The cells were washed again, and additional antibodies were added and incubation continued for 30 minutes. Cells were fixed in 1% paraformaldehyde and analyzed using a FACSCalibur flow cytometer (Becton Dickinson) equipped with both a 488-nm argon ion laser and a 635-nm red dye laser. Instrument setup was performed using FACSComp software (Becton Dickinson), and fluorescence channel calibration verified using QC3 beads (Flow Cytometry Standards, San Juan, PR).

To analyze FLAER binding or CD59 expression on lymphocyte subpopulations, gates were set using anti-CD3 or anti-CD 19. A combination of CD33 expression and right-angle light scatter was used to discriminate monocytes and granulocytes for gating. A minimum of 10,000 total events were collected and data displayed on a 1024 channel log scale. List mode data were analyzed using Cell Quest software (Becton Dickinson). To determine the precision of the FLAER test, the same experiments in 1 patient with PNH were repeated 5 times, and the coefficient of variation was calculated.

Results

Detection of GPI Anchor Proteins in Human and Murine Cell Lines

In control experiments, we found that FLAER bound to cells as well as native unlabeled aerolysin did, and flow cytometry using FLAER easily detected cells displaying GPI anchor proteins (data not shown). Confocal microscopy using FLAER showed that the murine cell line EL4 was heavily labeled with FLAER, whereas class f GPI anchor–negative mutant EL4 cells were only faintly visible Image 1. Flow cytometric analysis of the 2 murine cell lines after staining with FLAER was equally effective in detecting the absence of GPI-anchored proteins from the class f cells (data not shown). To determine whether FLAER could be used in a flow cytometric assay to distinguish cells lacking GPI anchor proteins, we made a 15% mixture of GPI anchor–negative cells (LD−) with the GPI anchor protein–replete line (LD−PIGA+) and stained the cell mixture with FLAER or with a fluorescent monoclonal antibody to CD59. GPI anchor expression is restored in the LD− cell line by stable transfection with an expression vector containing the PIGA cDNA.20 The flow cytometric histogram Figure 1 illustrates the ability of both FLAER and anti-CD59 to detect the 15% PNH population.

Image 1.

Image 1

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Glycosylphosphatidylinositol (GPI) anchor protein expression in murine cell lines. Confocal fluorescence microscopy view of the wild-type EL4 (A) and GPI anchor–negative mutant EL4 (Thy-1-f) (B) cells after staining with a fluorescent proaerolysin variant, FLAER.

Figure 1.

Figure 1

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Glycosylphcsphatidylinositol (GPI) anchor protein expression in human paroxysmal nocturnal hemoglobinuria cell lines. LD− cells were mixed with the GPI anchor-replete cell line LD− PIGA+ to establish a 15% population of GPI anchor protein deficient cells (solid line). Fluorescence intensity is displayed following staining with a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to CD59 (A) or FLAER (a fluorescent proaerolysin variant [B]). A pure population of LD− cells was used as a negative control (dotted line).

Detection of GPI Anchor Proteins in PNH

The GPI anchor deficit was assayed using anti-CD59 and FLAER in different leukocyte populations in a series of 10 healthy control subjects and 8 patients with PNH. PNH populations in granulocytes and especially monocytes were detected much more readily with FLAER than with anti-CD59 Table 1. The median channel fluorescence of CD59 expression on normal monocytes was 679 ± 55, while that of FLAER was 686 ± 36. In normal granulocytes, the median channel for CD59 was 795 ± 32, while that of FLAER was 747 ±31. While comparable fluorescent intensity was seen with anti-CD59 and FLAER in samples from control subjects, marked differences were observed in samples from patients with PNH. Anti-CD59 failed to detect a significant (>5%) PNH population among monocytes in 3 of 8 patients, while FLAER identified a minimum of 61% abnormal cells in these patients (Table 1). Dual parameter displays of anti-CD 14 vs anti-CD59 or FLAER clearly show that CD14-deficient monocytes were present but that FLAER, and not anti-CD59, was capable of identifying these populations Figure 2. Even when CD59-negative monocytes could be found, the median channel fluorescence between the PNH clone and the residual type I cells was much lower than that seen with FLAER; thus, discrimination between normal and PNH cells was more apparent with FLAER.

Table 1.

Comparison of GPI Anchor Protein Expression on Monocytes and Granulocytes of Patients With Paroxysmal Nocturnal Hemoglobinuria (PNH) Using Anti-CD59 and FLAER

Patient No. Monocytes
Granulocytes
Anti-CD59
FLAER
Anti-CD59
FLAER
%PNH Difference* %PNH Difference* %PNH Difference* %PNH Difference*
1   4 274  61 321 38 148 77 430
2 <1  –† 66 288 19 157 57 394
3 58 230 91 370 60 247 94 464
4 97   160‡ 93 318 97 242 94 508
5 63 155 99 338 80   226‡ 98 445
6  5 180 >99   388 87   218‡ 99   445‡
7 96   149‡ 99   331‡ 99 279 99   420‡
8 12 167 85 277 97 261 96 366
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FLAER, a fluorescent proaerolysin variant.

*

Difference in intensity between the median channels of PNH and residual type I cells.

†

No PNH population detected.

‡

No residual type I population detected; difference is that of intensity of PNH population and that of median of 10 healthy control subjects.

Figure 2.

Figure 2

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Expression of glycosylphosphatidylinositol (GPI) anchor proteins on monocytes using anti-CD59 and a fluorescent proaerolysin variant (FLAER) in a patient with paroxysmal nocturnal hemoglobinuria (PNH). Flow cytometric display of peripheral blood monocytes after staining with anti-CD14 APC and anti-CD59 FITC (A) or anti-CD14 APC and Alexa Fluor 488 conjugated mutant aerolysin (FLAER) (B). Although PNH cells can be recognized based on loss of CD14, these cells are not well discriminated with anti-CD59 but are easily separated with FLAER.

Improved separation between normal and PNH granulocytes also was observed with FLAER. Staining with anti-CD59 demonstrated that more than 90% of cells were GPI anchor deficient in 3 of 8 patients, while FLAER staining revealed that 6 of 8 patients had more than 90% abnormal cells (Table 1). In the remaining 2 patient samples, in which there were significant residual normal type I cells, FLAER detected a higher percentage of abnormal cells than anti-CD59 (Table I, patients 1 and 2). Most significantly, the discrimination between normal and PNH cells was greater using FLAER compared with anti-CD59 Figure 3, with an average difference of 434 log channels with FLAER compared with an average difference of 222 with anti-CD59.

Figure 3.

Figure 3

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Expression of glycosylphosphatidylinositol (GPI) anchor proteins on granulocytes using anti-CD59 and a fluorescent proaerolysin variant (FLAER) in a patient with paroxysmal nocturnal hemoglobinuria (PNH). Flow cytometric display of peripheral blood granulocytes after staining with anti-CD59 FITC (A) or FLAER (B). Although an abnormal population is demonstrated with both reagents, there is a larger difference in fluorescence intensity between the normal and PNH populations using FLAER. SSC, side scatter.

The size of the PNH lymphocyte population was relatively small with both anti-CD59 and FLAER, in agreement with previous findings.1 However, in cases in which a sizable PNH clone was identified, there was a tendency toward better discrimination between positive and negative populations with FLAER than with anti-CD59 (data not shown).

The FLAER-based flow cytometric assay not only is sensitive but it also is highly reproducible. Five replicate measurements of the percentage of PNH cells were made using cells from patient 1, and the median channel difference between normal and PNH cells was calculated on each of these samples. The coefficient of variation for the percentage of PNH cells detected by FLAER was 2.3% and 0.1% for monocytes and granulocytes, respectively (data not shown). Similarly, the coefficient of variation for the measured difference in intensity between the median channels of PNH cells and residual type I cells was 1.1% for monocytes and 1.2% for granulocytes (data not shown).

Use of FLAER to Detect Small PNH Populations in Aplastic Anemia

Although most patients with PNH will have a significant PNH population demonstrable with the use of monoclonal antibodies, there is an increasing need for the ability to detect minor PNH clones, particularly under circumstances in which assessment of the therapeutic response may be required. Furthermore, PNH frequently evolves from aplastic anemia, suggesting a pathophysiologic link between the 2 disorders.8,21–23 In fact, up to 50% of patients with aplastic anemia harbor a small PNH phenotype in 1 or more hematopoietic lineages.22 The improved resolution between normal and PNH cells suggests that FLAER should be superior in detecting minor PNH populations.

To investigate this possibility we first looked for the occurrence of FLAER-negative granulocytes in our series of healthy control subjects. Only 0.12% ± 0.14% of granulocytes were FLAER negative (range, 0.00–0.32) (data not shown), suggesting that FLAER could be used to detect abnormal populations on the order of 0.5% or more.

We next used 2-parameter flow cytometry using anti-CD59 to test the ability of FLAER to detect small (<10%) GPI anchor–deficient populations in 3 patients with aplastic anemia who were previously known to harbor a small PNH population. A representative example from 1 of these patients is shown in Figure 4. None of the control subjects (10 healthy subjects and 3 patients with myelodysplastic syndromes) displayed cells with a PNH phenotype (data not shown).

Figure 4.

Figure 4

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Detection of a small paroxysmal nocturnal hemoglobinuria population in a patient with aplastic anemia. Dual-color display of peripheral blood granulocytes after staining with anti-CD15 PE and anti-CD59 FITC (A) or anti-CD15 PE and a fluorescent proaerolysin variant (FLAER) (B), showing improved ability to detect the small glycosylphosphatidylinositol anchor protein–deficient population in this patient by using FLAER. PE, phycoerythrin.

Discussion

Traditional diagnostic assays for PNH, such as the Ham test, largely have been supplanted by flow cytometric assays for GPI anchor proteins. Although binding of CD59 or CD55 to erythrocytes is a simple assay to detect PNH cells, erythrocytes are not the best lineage to assay in PNH, particularly if quantitative estimates of the PNH clone are desired. Normal erythrocytes have a selective advantage compared with PNH erythrocytes from the same patient, since PNH erythrocytes are more susceptible to complement-mediated lysis. Furthermore, many patients who are screened for PNH have received erythrocyte transfusions, which also may contribute to underestimating the size of the PNH clone. As a result, PNH erythrocytes may be undetectable even when a significant PNH population is present. Thus, most flow cytometric screening assays attempt to detect GPI anchor protein expression on leukocytes, especially granulocytes and monocytes, since they most closely approximate the proportion of the stem cell defect in PNH and are not influenced by blood transfusions.1

No single antibody is optimal for detection of a GPI-anchor defect on all lineages. In contrast, we found the Alexa Fluor–labeled T253C/A300C (FLAER), a proaerolysin variant that can bind to GPI anchor proteins like native proaerolysin but cannot form channels, is an ideal reagent for the flow cytometric diagnosis of PNH. Since the GPI anchor is the major determinant of aerolysin binding, FLAER allows for the direct assay of GPI anchor expression on a variety of different cell types.

Interestingly, human erythrocytes proved to be an exception. Although we could measure a difference between normal and PNH erythrocytes using FLAER, it was not nearly as pronounced as the differences we observed with all of the other cell types we studied (data not shown). This may be because both normal and PNH erythrocytes contain large amounts of glycophorin, a protein shown to bind aerolysin weakly.18

There are situations where false-negative results could be obtained with the use of a single monoclonal antibody, such as CD59 or CD55, but not with FLAER. For example, rare congenital deficiencies of CD5924,25 and CD5526–28 unrelated to PNH have been described. In addition, certain cell lineages do not express specific GPI-anchored proteins or may appear to express these GPI anchor protein epitopes even when the GPI anchor is not present.

Our results comparing CD59 or FLAER binding with that of CD14 indicate that there are situations in which CD14-negative and FLAER-negative monocytes clearly continue to display CD59. The explanation for this is uncertain. It may be that there is transfer of soluble CD59 epitope to the surface of monocytes or that CD59 exists in both GPI-anchored and transmembrane forms. The former possibility has been demonstrated to occur in vitro, although not in vivo.29,30 The latter possibility has been described for other GPI anchor proteins,12,13 although not for CD59. Regardless of the mechanism, CD59, the most widely expressed GPI anchor protein on hematopoietic cells, is not an ideal target for assessing the GPI anchor defect in PNH monocytes. A second labeled antibody, such as anti-CD 14, is necessary. In contrast, FLAER binding is a much more accurate representation of the number of GPI anchor proteins on the cell surface.

FLAER also was more reliable than anti-CD59 for detecting the extent of the PNH clone in granulocytes. The intensity difference between positive and negative cells was more than 200 log channels greater with FLAER than with anti-CD59 (Table I). This greater sensitivity and reliability of detection with FLAER suggests that FLAER would be an improved reagent to detect small clones or to monitor levels of PNH clones in patients after therapy. Our studies with healthy control subjects suggest that detection of levels of 0.5% abnormal cells or more should be reliable. This may be particularly helpful for studying the close pathophysiologic relationship between PNH and aplastic anemia.

Our results show that for the routine diagnosis of PNH and for the detection of small PNH clones in aplastic anemia, flow cytometric assays using FLAER are superior to traditional flow cytometric assays using anti-CD59.

Acknowledgments

We thank Richard J. Jones, MD, for critical reading of the manuscript.

Supported in part by grant CA74990 from the National Institutes of Health, Bethesda, MD (R.A.B.), and by a grant from the Medical Research Council of Canada, Ottawa, Ontario (J.T.B.).

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