Clinical Evaluation of an Aerolysin-Based

Clinical Evaluation of an Aerolysin-BasedScreening Test for Paroxysmal NocturnalHaemoglobinuriaPietro Edmondo Peghini*and Jo¨rg FehrDivision of Haematology,Department of Internal Medicine,University Hospital Zurich,Zurich,SwitzerlandBackground:Recently,a toxin produced by Aeromonas hydrophila was demonstrated to bind directly to the glycosyl-phosphatidyl-inositol(GPI)anchor.After coupling it to afluorescent dye and applying it in fluorescence-activated cell scanning(FACS),this property was exploited to detect GPI-negative cells in the diagnosis of paroxysmal nocturnal haemoglobinuria(PNH).Methods:We used this reagent according to a very simple staining protocol followed by single-colour FACS and compared the results in patients with PNH and normal controls with those obtained with anti-body-mediated detection of cells lacking GPI-anchored proteins.Results:We observed very good concordance between the two methods,with correlation coefficients (R2)of quantified GPI-deficient cell populations ranging from0.952to0.969.The lower limit of detection was determined at0.50%GPI-negative cells,which was in the range obtained with double-colour stain-ing with antibodies(0.20–1.00%,depending on the antibody).A significant correlation was observed between the fraction of GPI-negative granulocytes and laboratory parameters of haemolysis,with the erythrocyte creatine having the best correlation(R2¼0.671,P<0.0001).Conclusions:Using this protocol,we were able to reliably diagnose PNH with a high sensitivity.The test allows the identification of GPI-negative granulocyte populations as small as0.5%.q2005Wiley-Liss,Inc. Key terms:paroxysmal nocturnal haemoglobinuria;FLAER;haemolysis;immunophenotypingFor many years the diagnosis of paroxysmal nocturnalhaemoglobinuria(PNH)was made by demonstrating thesusceptibility to complement-mediated lysis(1,2).Subse-quently,demonstration of the absence of glycosyl-phospha-tidyl-inositol(GPI)–anchored proteins from the cell surfaceusing antibodies andfluorescence-activated cell scanning(FACS)largely served to replace this approach(3–5).Typi-cally,only a fraction of the cells shows the PNH immuno-phenotype,and it is the demonstration of two cell popula-tions,one positive and one negative in GPI-anchored pro-teins,that is diagnostic for the disease.In different cellpopulations,various antigens are investigated.Aerolysin,the toxin of the bacterium Aeromonashydrophila,binds directly to the GPI anchor.It is secreted as an inactive protoxin,proaerolysin,that isconverted to the active form,by various mammalian pro-teases,through proteolytic removal of a C-terminal pep-tide(6,7).Aerolysin has been used in diagnostic proce-dures to demonstrate the resistance of PNH erythrocytesto aerolysin and to enrich for GPI-negative PNH cells(8,9).Also,two point mutations were introduced toobtain a protein that still binds GPI upon activation butlacks lytic activity.By coupling this mutant proaerolysin to afluorescent marker(Alexa Fluor488),a reagent (FLAER)was produced that stains cells containing GPI proteins but not PNH cells lacking GPI(10).Because this reagent detects the GPI anchor itself,it can be used to investigate all cell types,irrespective of the proteins normally expressed,except erythrocytes,which do not express the necessary activating proteases.In contrast to immunophenotyping,where several antibodies must be employed to investigate different cell populations,FLAER can provide the same information in a single reaction. We used FLAER in whole blood according to a very simple and fast staining protocol as a screening test for the diagnosis of PNH.We determined the lower limit of detection with this simple procedure and compared the results with those from conventional immunophenotyp-ing performed in parallel.In addition,results obtained *Correspondence to:Pietro E.Peghini,Division of Haematology,Uni-versity Hospital Zurich,Ra¨mistr.100,CH-8091Zurich,Switzerland.E-mail:pietro.peghini@usz.chReceived23August2004;Accepted5April2005Published online10June2005Wiley InterScience(www. ).DOI:10.1002/cyto.b.20059Cytometry Part B(Clinical Cytometry)67B:13–18(2005)q2005International Society for Analytical CytologyF IG .1.Demonstration of GPI-negative cells by antibodies and FLAER.A representative example is shown with FACS results for a patient and for a normal control.Parameters measured are as indicated.Gating was performed in a forward versus side scatter plot (Gc,granulocytes;M,monocytes).Antibodies were coupled with fluorescein isothiocyanate (FITC)or phycoerythrin (PE).Antibodies used are indicated on the axes.14PEGHINI AND FEHRwith FLAER were correlated with known parameters of haemolysis.MATERIALS AND METHODSDetection of GPI Anchor by FLAER AssayFLAER was obtained from Protox Biotech (Victoria,Canada).It was stored at 10À6M in phosphate buffered saline (PBS)at À208C.To 25m l of whole blood,anticoa-gulated with ethylenediaminetetraacetic acid or heparin,5m l of the FLAER solution was added,mixed gently,and incubated for 10to 15min in the dark at room tempera-ture.After addition of 500m l of erythrocyte lysing reagent (0.829%NH 4Cl,0.1%K 2HCO 3,and 0.0037%ethyl-enediaminetetraacetic acid in distilled water),samples were measured in parallel with a normal control by sin-gle-colour FACS analysis in channel 1,corresponding to green fluorescence,on a FACScalibur (Becton Dickinson,Basel,Switzerland).Leukocyte subpopulations were gated on a forward versus side scatter plot.A normal control was always stained in parallel.The threshold fluorescence for negative versus positive cells was set where the main peak of this normal control intersected the x-axis.ImmunophenotypingTo 100m l of whole blood,antibodies (Becton Dickin-son)were added at the dilution suggested by the manu-facturer and incubated for 15to 30min at room temper-ature in the dark.Erythrocytes were lysed with 2ml of lysing reagent and leukocytes washed once with 3ml of standard PBS and resuspended with 500m l of PBS.Leu-kocyte subpopulations were identified on a forward ver-sus side scatter plot,which allowed gating for granulo-cytes and monocytes.CD55-negative cells were investi-gated in granulocytes and monocytes in this manner.This test therefore was a single-colour fluorescence experi-ment.For dual-colour fluorescence analysis of CD16,CD24,and CD66b,a monoclonal antibody with a differ-ent fluorescent label (CD13/phycoerythrin)was added to the assay to improve the identification of myelomono-cytic cells.Thus,the investigated cell populations,granu-locytes and monocytes,were identified by virtue of their positivity for CD13and their light scatter properties.CD59staining was performed with CD3/phycoerythrin,which allowed elimination of T cells that could have con-taminated the gates for granulocytes and monocytes based on forward versus side scatter characteristics.CD14was investigated with dual-colour fluorescence analysis on monocytes.A CD64/phycoerythrin-labelled monoclonal antibody served to discern monocytes from lymphocytes contaminating the monocyte gate in for-ward versus side scatters.Lymphocytes were not ana-lysed.We investigated CD16,CD66b,CD24,CD59,and CD55on granulocytes and CD14,CD59,and CD55on monocytes.A normal control was always stained in parallel.The threshold fluorescence for negative versus positive cells was set between the two,readily visible populations in samples with an obvious PNH component.In cases with-out two clear populations,the threshold was set below the dot cloud in the normal control in such a manner that only events clearly separated from this dot cloud were counted as nonfluorescent.Statistical AnalysisLinear correlation and calculation of the correlation coefficient (R 2)were performed with StatView software.RESULTSDemonstration of GPI-Negative CellsGPI-deficient cells were sought by using antibodies as indicated in Materials and Methods and FLAER in sevenTable 1Percentage of GPI-Negative Cells in Granulocytes and Monocytes With FLAER and Various Antibodies*Patient no.Diagnosis %Negative granulocytes %Negative monocytesPNH clone FLAER CD16CD24CD55CD59CD66b FLAER CD14CD55CD591PNH 40292522242643373231Yes 2PNH 981001009910099961009996Yes 3AA/PNH 101199991091234Yes 4MDS0.3 1.50.1 1.90.10.10.30.3 1.2 1.5No 5MDS/PNH516753616741633759Yes 516869535465415047Yes 27372827222621321830Yes*Values were rounded to the nearest integer.The two values missing are due to technical errors.Patient 5was measured three times on different occasions during treatment with antilymphocyte globulin and ciclosporin.AA,aplastic anemia;MDS,myelodys-plastic syndrome.Table 2Correlation Coefficients Between FACS Mediated byFluorescent Antibodies or FLAER in Granulocytes or Monocytes*R 2FLAER GcFLAER MonoGc CD160.959Gc CD240.950Gc CD550.9680.956Gc CD590.9690.901Gc CD66b 0.957CD140.918*Excellent correlation was observed,demonstrating compa-rable accuracy of the two methods in quantifying PNH cells.Gc,granulocytes;Mono,monocytes.15AEROLYSIN-BASED TEST FOR PNHmeasurements in five patients and seven normal con-trols.Figure 1shows the FACS results of patient 5and the respective normal control.Diagnoses were PNH in two,myelodysplastic syndrome (MDS)with a PNH clone in one,MDS without a PNH clone in one,and aplastic anaemia with a PNH clone in 1patient (Table 1).Rele-vant GPI-negative cell populations were observed in four of five patients and in none of seven controls (controls not shown).These results,expressed as dichotomousvalues of PNH clone present or PNH clone absent,were completely concordant between the two methods (Table 1,last column).Correlation Between FACS Mediated by AntibodyVersus FLAER Fourteen measurements of antibody-mediated FACS were performed in parallel with FLAER analysis.The nonfluorescent fraction was determined for each investi-gation and the correlation was determined bylinearF IG .2.Regression plots of nonfluorescent fractions from FACS medi-ated by FLAER versus fluorescent monoclonal antibodies.Representa-tive examples of regression plots are shown.Values on the axes repre-sent percentages of nonfluorescent cells.FLAER is always on the verti-cal axis,and monoclonal antibodies are as indicated on the horizontal axis.Correlation coefficients (R 2)are given in the plots.Gc,granulo-cytes;Mono,monocytes.Table 3Fluorescence-Negative Events in Normal Controls by FLAERAnalysis and Antibody-Mediated FACS*FLAERCD16CD66b CD24CD59CD55n 5077777Mean 0.049 2.4510.1130.2470.143 1.053Minimum 0.000.850.040.000.000.07Maximum 0.488.310.260.990.573.62*Values are percentages of nonfluorescent granulocytes gated by forward versus sidescatter.F IG .3.Percentile plot of fluorescence-negative events in FLAER ver-sus fluorescent monoclonal antibody-mediated FACS in normal con-trols.Values on the vertical axis are percentages of fluorescence-nega-tive events.Percentiles are indicated on the horizontal axis.Only the granulocyte (Gc)gate was investigated.A:FLAER versus antibodies used in a dual-colour FACS experiment.B:FLAER versus CD55stain-ing that was performed as a single-colour FACS experiment.16PEGHINI AND FEHRregression analysis.Correlation coefficients (R 2)and regression plots are presented in Table 2and Figure 2,respectively.Excellent correlations between PNH popu-lations detected by the two methods were observed,demonstrating a comparable accuracy of the two meth-ods for the quantification of PNH cells.Lower Limit of DetectionFor analysis of the lower limit of detection,only granu-locytes were investigated because they represent the largest fraction and were more easily gated in the for-ward versus side scatter plot.Fifty normal control sam-ples were assayed by FLAER and seven by immunophe-notyping.Values between 0%and 0.48%FLAER-negative events were counted in the granulocyte gate.The values observed with fluorescent antibodies were in the same range in double-colour FACS results,as presented in Table 3and by a percentile plot in Figure 3A.However,we performed the CD55staining as a single-colour fluo-rescence assay,and a higher background was observed (0.07–3.62%;Fig.3B).In addition,in CD16staining,higher values were measured in normal controls,but this was due to the absence of CD16from normal eosi-nophil granulocytes.In 50normal controls,the highest value of fluorescence-negative events with FLAER was 0.48%.Therefore,a lower limit of detection can be set at 0.5%.FLAER Correlates With Haemolytic ActivityThe fraction of FLAER-negative cells as a percentage correlated only very weakly with the parameters of hae-molysis LDH,reticulocyte count,and bilirubin.A weak but definitely better correlation was found with the hae-molysis parameter of erythrocyte creatine (R 2¼0.671,P <0.0001;Fig.4)(11).DISCUSSIONPNH enters the differential diagnosis in every case of Coombs-negative haemolysis.In addition,it has recently been shown that patients with myelodysplastic syndrome carrying a PNH clone,even if it is very small,respond more readily to immunosuppressive treatment (12).Therefore,although PNH is a rare disease,it is quite fre-quently investigated,and a simple,reliable screening test with sufficient sensitivity for small PNH clones would be of benefit.Haemolysis in PNH arises from a lack of complement inhibitors from the surface of erythrocytes.This is due to the inability of these cells to synthesize the GPI anchor.Until a few years ago,diagnosis had to rely on indirect methods that demonstrated increased suscepti-bility to complement-mediated lysis (1,2)or the lack of GPI-anchored proteins by immunological methods (3–5).With aerolysin,the toxin of A.hydrophila ,which specif-ically binds to the GPI anchor,a more straightforward diagnostic approach has become possible (10).We have shown that the diagnosis of PNH can be made reliably using this reagent with a very simple and rapid staining protocol and single-colour fluorescence detection.Nonfluorescent events in the FACS analysis of normal controls were always less than 0.5%of peripheral granulocytes.Therefore,the test has a lower limit of detection for PNH clones as small as 0.5%.A comparable performance was obtained with monoclonal antibodies only with dual-colourstaining.F IG . 4.Correlation between the number of FLAER-negative cells and parameters for haemolysis.Linear correlation plots compare percentages of FLAER-nega-tive cells with known parameters of haemolysis.The best correlation was observed with erythrocyte creatine (ec crea;P <0.0001)(11),although all correlations were rather weak.Gc,granulocytes;LDH,lactate de-hydrogenase.17AEROLYSIN-BASED TEST FOR PNHProbably by counterstaining the cell population of interest with an antibody labelled with a secondfluores-cent dye in a FLAER experiment,the lower limit of detection for GPI-negative cells could be decreased. However,this was not the goal of the present method, which was deliberately designed as a rapid and simple screening test for routine analysis.Lymphocytes could not be investigated with the present method because the gating strategy did not per-mit separating them reliably from remaining non-lysed erythrocytes and probably erythrocyte ghosts.Staining with an antibody carrying a secondfluorescent label would likely solve this problem in a setting where the investigation of lymphocytes was important.Moreover, erythrocytes are not amenable to analysis by FLAER because they lack the proteases necessary for proteolytic activation of proaerolysin(10).Because the percentage of FLAER-negative cells corre-lated to a certain degree with haemolytic activity,it can be used as a follow-up parameter to estimate disease severity and response to treatment.In conclusion,by using this very rapid FLAER-based protocol for FACS,we were able to reliably detect GPI-negative cell populations in patients carrying a PNH clone.PNH clones as small as0.5%were readily detected.Because of its simplicity and correlation to hae-molytic activity,this test also lends itself to serial follow-up of the course of PNH.ACKNOWLEDGMENTSWe thank Mrs.Ruth Kalberer,Mrs.Evelyne Giabbani, Mrs.Michelle Handschin,Mrs.Francesca Bianda,and Mrs.Leta Fuchs for excellent technical assistance in FACS.LITERATURE CITED1.Hartmann RC,Jenkins DE.Sugar-water test for paroxysmal noctur-nal hemoglobinuria.N Engl J Med1966;275:155–157.2.Hartmann RC,Jenkins DE,Arnold AB.Diagnostic specificity ofsucrose hemolysis test for paroxysmal nocturnal hemoglobinuria.Blood1970;35:462–475.3.van der Schoot CE,Huizinga TW,ET vtV-K,Wijmans R,Pinkster J,dem Borne AE.Deficiency of glycosyl-phosphatidylinositol-linked membrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria,description of a new diagnostic cytofluorometric assay.Blood1990;76:1853–1859.4.Hall SE,Rosse WF.The use of monoclonal antibodies andflowcytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria.Blood1996;87:5332–5340.5.Schubert J,Alvarado M,Uciechowski P,et al.Diagnosis of paroxys-mal nocturnal haemoglobinuria using immunophenotyping of peripheral blood cells.Br J Haematol1991;79:487–492.6.Howard SP,Buckley JT.Activation of the hole-forming toxin aeroly-sin by extracellular processing.J Bacteriol1985;163:336–340.7.Garland WJ,Buckley JT.The cytolytic toxin aerolysin must aggre-gate to disrupt erythrocytes,and aggregation is stimulated by human glycophorin.Infect Immun1988;56:1249–1253.8.Mukhina G,Buckley T,Brodsky RA.A rapid spectrophotometricscreening assay for paroxysmal nocturnal hemoglobinuria.Acta Hae-matol2002;107:182–184.9.Brodsky RA,Mukhina GL,Nelson KL,Lawrence TS,Jones RJ,Buck-ley JT.Diagnosis of paroxysmal nocturnal hemoglobinuria(PNH) and selection of small PNH populations using a novel GPI-anchor binding toxin,aerolysin.Blood1998;92:472A.10.Brodsky RA,Mukhina GL,Li S,et al.Improved detection and char-acterization of paroxysmal nocturnal hemoglobinuria usingfluores-cent aerolysin.Am J Clin Pathol2000;114:459–466.11.Fehr J,Knob parison of red-cell creatine level and reticulo-cyte count in appraising the severity of hemolytic processes.Blood 1979;53:966–976.12.Dunn DE,Tanawattanacharoen P,Boccuni P,et al.Paroxysmal noc-turnal hemoglobinuria cells in patients with bone marrow failure syndromes.Ann Intern Med1999;131:401–408.18PEGHINI AND FEHR。