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From www.bloodjournal.org by guest on August 10, 2017. For personal use only. The Use of Monoclonal Antibodies and Flow Cytometry in the Diagnosis Paroxysmal Nocturnal Hemoglobinuria of Bv Sharon E. Hall and Wendell F. Rosse We have characterized the erythrocytes, granulocytes, and platelets of 54 patients with paroxysmal nocturnal hemoglobinuria (PNH) with antibodies to glycosylphosphatidylinositol-anchored proteins (anti-CD55, anti-CD59, and anti-CD161 and flow cytometry to establish the usefulness of this technique in the diagnosis of this disorder. All patients demonstrated either completely (PNH 111) or partially (PNH I11 deficient red cells and granulocytes. Anti-CD59 best demonstrated PNH II red cells, which were present in 50% of the patients. The proportion of abnormal granulocytes was usually greater than the proportion of abnormal red cells; 37% of the patients had >80% abnormal granulocytes.Anti-CD55 did not delineate the erythrocyte populations as well as did anti-CD59. Either anti-CD55 or anti-CD59 could be used equally well toanalyze granulocytes; anti-CD16 did not demonstrate cells of partial deficiency. Platelets could not be used fordetailed analysis asthe normal and abnormal populations werenotwell distinguished. Flow cytometry of erythrocytes using anti-CD59 or of granulocytes using either anti-CD55 or anti-CD59 provides the most accurate technique for the diagnosis of paroxysmal nocturnal hemoglobinuria; it is clearly more specific, more quantitative, and more sensitive than the tests for PNH that depend upon hemolysis by complement (the acidified serum lysis [Ham] test, the sucrose lysis test, and the complement lysis sensitivity [CLS] test). 0 1996 by The American Society of Hematology. P and unseparated granulocytes of S4 patients, purified granulocytes of 39 patients,andplateletsof 18 patientswere tested by flow cytometry.Samples of bloodfromhealthy adult volunteerswere analyzed concurrently to determine normal values. AROXYSMALnocturnalhemoglobinuria (PNH) is characterized by the deficiency, absolute or partial, of all proteins anchored to the membrane by the glycosylphosphatidylinositol (GPI) anchor’”; at least 1S of these proteins have been shown to be lacking on the abnormal blood cells of patients with this disease. In the past, the diagnosis of PNH has relied on the demonstration of the effect of the absence o f t w oof these proteins, CD59 (membrane inhibitor comof reactive lysis[MIRL]? protectin,s,6 membrane attack plex inhibiting factor [MACIF],’ etc). and CDSS (decay accelerating factor [DAF],’ which results in the abnormal sensitivity of the red cells to the lytic action of complement.”“’ In the present study, we report the results of the use of monoclonal antibodies (MoAbs) against CDSS, CDS9, and CD16 (the Fc,receptor 111) in the analysis byflow cytometry of the red cells, granulocytes, and platelets of 54 consecutive PNH patients. We demonstrate the ability of this methodolof ogy to distinguish the presence of even small populations blood cells partially or totally deficient in these proteins and to assess accurately the proportion of cells in these populawe establish for the first time the tions. By this analysis, optimum use of this methodology in the diagnosis of this disease. MATERIALS AND METHODS Muterials The MoAb 1H4, specific for CD55, was produced in our laboratory. An MoAb to CD59, 10G10, was the gift of Dr Marilyn Telen (Duke University, Durham, NC). An MoAb to CD16 (3G8) was the gift of Dr Mark Cunie. Mouse anti-human glycophorin A antibody labeled withR-phycoerythrin(R-PE)waspurchasedfromPharmingen(SanDiego,CA).AnMoAb,CD13directly labeled with R-PEwasobtainedfromSouthernBiotechnologyAssociates,Inc (Birmingham, AL). Nonreactive MoAb P3 with random specificity was used as a negative control. Fluorescein-conjugated goat F(ab’ anti-mouse IgG(GAM-FITC) was purchased from Biosource International (Camarillo, CA). Normal goat serum and Dulbecco’s phosphate-bufferedsaline(DPBS)werepurchasedfromGIBCOBRL (GrandIsland, NY). Humanserumwasobtainedfromnormal healthy group AB volunteers. Ortho-mune lysing reagent was purchased from Ortho Diagnostic Systems Inc (Raritan, NJ). Dextran T500 was obtained from Pharmacia Biotech Inc (Piscataway, NJ). LymphocyteSeparationMedium(LSM) was purchasedfrom Organon Teknika Corporation (Durham, NC). The buffer used in the l b w cytometry assays was veronal-buffered saline (GVB-EDTA), composed of 0.005 mol/L sodium barbital, 0.15 m o l L NaC1. 0.1o/r gelatin, 0.01.5 mol/L EDTA, pH 7.5. Patients Preparatiort ?f Cells for Flow Cytometry The blood of S4 patients (23 men, 3 I women) who had or were suspected of having the diagnosis of PNH was tested. The patients ranged in age from 1 1 to 65 years (mean 34 years). Erythrocytes Venous peripheral blood was drawn from patients with PNH or suspected of having PNH and normal controls into EDTA-containing tubes. For whole blood analysis, 400 p L of whole blood was removed and 100 pL was placed in each of four tubes. Two milliliterh of Ortho-mune lysing reagent was added and incubated for 10 minutes. The tubes were centrifuged at 1500rpm for 5 minutes, and the pellet washed once in GVB-EDTA. The pellet was then resuspended to 100 pL in GVB-EDTA. Erythrocytes were washed three times to 1 X IO’/mLin GVB-EDTA. in GVB-EDTAandresuspended Granulocytes were purified from whole blood by diluting the blood with an equal volume of DPBS and layering the mixture over an equal amount of LSM. The mixture was spun at 2000 rpm for 30 minutes. The granulocytes were further purified by sedimentation of the erythrocytes with Dextran on ice for 45 minutes. The granulocytes were washed in GVB-EDTA and resuspended to 5 X 107/mL in GVB-EDTA. Platelets were obtained by centrifuging whole blood From the Division of Hematology-Oncology,Department of Medicine, Duke University Medicul Center, Durham. NC. Submitted June 9, 1995; accepted February 16, 1996. Address reprint requests to Wendell F. Rosse, MD, Box 3934. Duke University Medical Center, Durham, NC 27710. The publication costs of this urticle were defrayed in part by page charge puyment. This urticle must therefore be hereby marked “advertisement” i n accordance nith I 8 U.S.C. section 1734 solelv to indicate this fact. 0 1996 by The American Society of Hematologv. 0006-4971/96/8712-0002$3.00/0 5332 Blood, Vol 87,No 12 (June 15), 1996: pp 5332-5340 From www.bloodjournal.org by guest on August 10, 2017. For personal use only. 5333 DIAGNOSIS OF PNH BY FLOW CYTOMETRY at 800 rpm for 15 minutes and removing the platelet-rich plasma. The platelet-rich plasma was then centrifuged at 1500 rpm for 5 minutes, and the platelet pellet was washed twice and resuspended to 1 X 108/mLin GVB-EDTA. Q 2 140 Immunophenotype Analysis Q Single color flow analysis was performed by using a two-step method. One hundred microliters of the various cell suspensions were incubated with 100 pL of each MoAb and P3 for 30 minutes at room temperature. The cells were then washed twice with GVBEDTA and resuspended to 100 pL volume.A 10-pL mixture of GAM-FITC, normal goat serum, and group AB normal human serum (ratio 1:1:2.5) was added and incubated for another 30 minutes at room temperature, followed by two washes in GVB-EDTA. The pelleted cells were resuspended in 1 mL of buffer and analyzed on an Ortho Cytoron Absolute (Ortho Diagnostic Systems Inc). Cell types were determined by their forward and right scatter. Two-color analysis was performed as above except that MoAbs to specific cell markers and directly labeled with R-PE were used at the recommended manufacturer’s dilutions as a third step. Color compensation for crossover of fluorescence signals between the two detectors was adjusted for optimal analysis. Complement Lysis Sensitivity Test (CLS) A modified version of the original CLS test of Rosse and Dacie’ was used to compare erythrocyte populations to flow cytometry. The reaction volume was reduced to 375 pL withthe proportions of erythrocytes, complement, and buffer staying the same. PNH cells give complex curves that are analyzed to determine the three different PNH cell populations. RESULTS Erythrocytes Normal erythrocytes. The erythrocytes from 44 normal donors were examined with anti-CD55 and those of 49 normal donors with anti-CD59 (Table 1).The mean fluorescence was greater with anti-CD59. Cells not reacting with the antibody used (unstained cells) were present in small numbers in each case. Erythrocytes from two normal donors were maintained at 4°C for up to 25 days to determine the stability of the detection of CD59. Six replicate samples were analyzed at each point for percentage of cells bearing CD59 and for the mean fluorescence obtained understandardized conditions. The percentage of cells bearing CD59 remained stable in time (range 98.3% to 99.8% and 98.4% to 99.7%, respectively) Table 1. Analysis of Normal Erythrocytesby Flow Cytometry and Anti-CD55 and Anti-CD59 ~~ No. of samples Mean fluorescence (channel) Unstained cells No. of samples’ Mean percentage Range (%) ~ Anti-CD55 Anti-CD69 44 76.8 2 12.1 49 137.5 2 21.2 20 (45) 0.23 2 0.34 0-1.4 41 (83.7) 0.31 -c 0.39 0-1.6 * Values in parentheses are percentages. 0 g 120 v) 3 ii c 100 m 80 l 0 5 I 1 , l ~ 15 20 Days in Storage 10 ! I 25 I 30 Fig 1. The mean fluorescence channel of normal cells (open symbols) and PNH I andlor PNH II cells (closed symbols) during storage in plasma at 4°C. as did the standard deviation of the determination (range 0.03% to 0.94%). The meanfluorescencedecreasedwith time (Fig 1). PNH erythrocytes. The washed erythrocytes from 54 patients with the diagnosis of or suspected of having the diagnosis of PNH were examined by flow cytometry with antiCD59 and anti-CD%. Using anti-CD59, three types of cells could be distinguished (Fig 2): cells with nearly normal expression of GPI-anchored protein (PNH I cells), cells with no detectable expression of GPI-anchored proteins (PNH111 cells), and cells with intermediate expression of GPI-anchored proteins (PNH I1 cells). Cells of intermediate expression occurred either as a discrete population(Fig2c), as “transition” between PNH 111 and PNH I cells (Fig 2d), or as an indistinct major population (Fig 20. The mean fluorescence using anti-CD59 of these populations is shown in Fig 3. An intermediate population was more readily seen when anti-CD59 was used, in part because the mean fluorescence of the PNH I cells is greater than with anti-CD%. The proportion of cells in each erythrocyte population was analyzed and the results for anti-CD59 are shown in Table 2. The pattern of populations of PNH erythrocytes for 54 patients is shown in Table 3. In 38 patients, the results of flow cytometry analysis were compared to the results of the CLS test. The results were concordant in 25 of 38 patients (65.8%) in that both identified the same populations, although the proportion of cells in the populations may not have been entirely similar. The CLS test did not detect a very small abnormal population of cells in one patient, whereas no patients tested had abnormal cells by CLS and none by flow cytometry. In the other 12 patients (31.2%), the CLS test did not identify correctly the degree of abnormality (PNH I v PNH I1 v PNH 111) or did not identifysmall populations (particularly small populations of PNH I1 cells). To determine the reproducibility and stability of the determinations of CD59 on PNH erythrocytes, the blood of three patients was maintained at 4°C for up to 25 days and was tested at intervals, analyzing six replicate samples at each From www.bloodjournal.org by guest on August 10, 2017. For personal use only. HALL AND ROSSE 5334 Normal Fig 2. Histograms of fluorescence of erythrocytes using anti-CD59. (a) Normal erythrocytes and inert antibody controls (P3). (b through e) Representative patterns from selected patients. (b) PNH l and PNH 111 cells; (c) discrete populations of PNH 1, PNH II, and PNH 111 cells; (dl discrete PNH I and PNH II cells, indistinct (transition) PNH II cells; (e) nearly entirely PNH 111 cells; (f) primarily PNH II cells with indistinct PNH I and PNH 111. . point for percentage of abnormal (PNH I1 andor 111) cells and the mean fluorescence channel of each population. The fraction of abnormal cells diminished with time (Fig 4); the mean fluorescence of PNH I and PNH I1 cells diminished at the same rate as that of normal cells (Fig 1). Analysis of Granulocytes Normal granulocytes. The granulocytes, separated or unseparated, from normal donors were examined with anti- CD55 and anti-CD59 (Table 4). In many donors, unstained cells were found; a larger proportion of these cells were found when unseparated granulocytes were examined, particularly when anti-CD59 was used in analysis. PNH granulocytes. The separated and unseparated granulocytes of 35 patients with PNH or suspected of having PNH were analyzed using anti-CD%, anti-CD59, and antiCD16 (Fig 5a through f). The cells could be divided into populations of PNH I, PNH 11, and PNH 111, using criteria 1; 20 .c, 15 c .-a, Y I - 1 Il L e 3 5 Fig 3. The mean fluorescence channel of anti-CD59 on erythronormal erythrocytes. I:, cytes !...; ,inert antibody(P3) controls; 0,PNH I cells; B B , PNH II cells (discrete population); m, PNH I1 cells (transition population); m, PNH 111 cells. z I 0 50 100 Mean Fluorescence Channel I I -1 150 From www.bloodjournal.org by guest on August 10, 2017. For personal use only. DIAGNOSIS OF PNH BY FLOW CYTOMETRY 5335 Table 2. Percentage of Abnormal Cells in Each of the Three PNH Populationsin Erythrocytes and Isolated Granulocytes ~________ Anti-CD551 PNH Granulocytes Percent ~ (Anti-CD59) ~~ Erythrocytes I 0-20 20-40 40-60 60-80 80- 100 Total 9 (16.7) 15 (27.8) 8 ( 14.8) 15 (27.8) 7 (13.0) 54 (100) PNH II PNH Ill PNH I PNH II PNH Ill 15 (26.8) 3 (5.4) 1 (1.8) 12 (21.4) 12 (21.4) 11 (19.6) 12 (21.4) 7 (12.5) 54 (96.4) 14 (40.0) 5 (14.3) 6 (17.1) 2 (5.7) 4 (11.4) 31 (88.6) 6 (17.1) 1 (2.9) 1 (2.9) 2 (5.7) 2 (5.7) 7 (20.0) 8 (22.9) 14 (40.0) 33 (94.3) - 2 (3.6) 21 (37.5) 1 (2.9) 9 (25.7) Values in parentheses are percentages. similar to those used for analyzing red cells. The separation of the populations and the ability to analyze accurately their percentage was greater with separated than unseparated cells. The mean fluorescence of the cells in each population is shown for separated cells in Fig 6. The proportion of cells in each population for 35 patients is shown in Table 2. The pattern of populations of PNH granulocytes for 35 patients is shown in Table 3. The relationship between the proportion of cells in each population to the proportion of cells in the corresponding red cell population is shown in Fig 7. In 23 of 35 cases (65.7%), the proportion of abnormal granulocytes was greater than the proportion of abnormal red cells, in 7 cases (20.0%) not significantly different, and in 5 cases (14.3%), it was less. Anti-CD16 also distinguished PNH granulocytes but did not distinguish populations of intermediate content (PNH I1 cells). Normal and PNH Platelets Normal and PNH platelets were testedwith anti-CD55 and anti-CD59. The amount of both proteins detected on the surface of normal cells was small with the result that the populations of cells in PNH patients were not well distinguished. DISCUSSION by van der Schoot and associates in 1990 in the analysis of granulocytes withapanel of antibodies including antiCD16." These studies were confirmed by Schubert et all2; in both studies, the totally deficient and normal populations of cells were found in all blood elements but partially deficient cells were not identified. Red cells of intermediate abnormality were identified in flow cytometry by Rosse et all3 and Shichishima et al,'4s'5 and, in both studies, were compared with the results obtained by the CLS test. More recently, Kwong et all6 have indicated other antibodies that may be used in detecting GPI-deficient cells in PNH. In none of these studies were the antibodies used compared in a large number of patients to establish the optimal conditions and techniques that should be used in the diagnosis of PNH and characterization of the cells in this disorder. In the present study, we have analyzed the cells of 54 patients with PNH to establish the optimum conditions for using flow cytometry in the diagnosis of the disorder. We have found that the analysis of erythrocytes is best carried out using anti-CD59. The mean fluorescence obtained with the GPI-positive cells is sufficiently great as to be readily distinguished from the GPI-negative cells; this was not the case with anti-CD55 (anti-DAF). The mean fluorescence of the GPI-positive PNH I cells in PNH was significantly less 50 I I T The use of MoAbs to GPI-linked proteins and flow cytometry in the definition of the defect in PNH was firstproposed Table 3. Patterns of Populations of Erythrocytes and Granulocytes in the Blood of Patients with PNH Populations* Erythrocytes (Anti-CD59) Granulocytes (Anti-CD55) PNH I + II PNH I + II + 111 PNH I + (11) 111 PNH I + 111 PNH II PNH II + 111 PNH Ill PNH (I + II) + 111 + 15 (27.8) 6 (11.1) 27 (50) 2 (3.6) 4 (7.1) 1 (2.9) 2 (5.7) 1 (2.9) 18 (51.4) 1 (2.9) 2 (5.7) 1 (2.9) 9 (25.7) Values in parentheses are percentages. * Parentheses indicate that the population is indistinct or transitional. 1 I" 0 5 10 15 Day 20 25 Fig 4. Changes in the percent of abnormal (PNH II [open rymbolsl andlor PNH 111 [dosedsymbolsl) cells with time instorage in plasma at 4°C. Each point is the mean of six replicate doterminatlons; l SD is indicated by the bars associated with it. From www.bloodjournal.org by guest on August 10, 2017. For personal use only. HALL AND ROSSE 5336 Table 4. Analysis of Isolated and Nonisolated Normal Granulocytes by Flow Cytometry and Anti-CD55 and Anti-CD59 Separated'Granulocytes AntiCD55 No. of samples Mean fluorescence (channel) Unstained cells No. of samples+ Mean percentage Range (%) 15 119.7 't 7.6 6 (40) 0.15 't 0.25 0-0.9 Anti-CD59 16 132.1 2 32 9 (56) 0.13 2 0.19 0-0.6 Unseparated Granulocytes AntLCD55 50 136.5 't 10.7 47 (94) 0.49 't 0.74 0-3.6 Anti-CD59 53 160 z 39 51 (96.2) 2.89 't 4.12 0-24.6 Values in parentheses are percentages. than that of normal cells, a result also obtained by Shichishima et al"; this suggests that the PNH I cells of PNH may not be entirely normal in their content of GPI-linked proteins. A very small number of cells not reacting with the antibodies was found in the blood of a few normal control subjects. Analysis by two-color cytometry as well as repeated analyses from the same patient suggested that these did not represent a small population of PNH-like cells. Their presence did, however, set the lower limit of detection of a PNH population at about 1% of the cells. Red cells of intermediate expression of GPI-dependent proteins were more readily identified using anti-CD59 than anti-CD55 These appeared to occur in three patterns: a distinct population of cells (17 patients), a continuum of cells between the PNH I and I11 cells (8 patients) and as a majority of the cells with deficient and nearly normal cells at each end of the spectrum (2 patients). All in all, 27 of 54 patients were found to have cells of intermediate expression. These findings correspond well with the findings using special tests of complement sensitivity and cell separation." However, when compared with the complement lysis sensitivity test alone, PNH I1 cells weremorereadilyidentified byflow cytometry. These studies demonstrate the degree of variability of the determination of the fraction of PNH cells in each population and the amount of CD59 on the membrane (by determination of the mean fluorescence channel) by replicate sampling. In addition, they show that the amount of CD59 onnormal cells and PNH I and I1 cells gradually decreases as the cell is stored; this protein may be digested by enzymes in the plasma or may dissociate from the membrane, as has been demonstrated for GPI-linked proteins." They also demonstrate that the fraction of abnormal cells consistently falls over time; the reason for this is not apparent. These two factors suggest that analysis is best performed with samples as fresh as possible. The analysis of granulocytes for the deficiency of GPIdependent proteins can also be used to diagnose PNH.".l6 AI1 three antibodies used (anti-CD59, anti-CD%, and antiCD16) readily demonstrated the deficiency of GPI-dependent proteins in some of the cells of all patients andthe proportion detected by each was approximately the same. Fig 5. Histograms of fluorescence of granulocytesusing anti-CD59 and FITC-labeled antiIgG. (a) Normal granulocytes; (b) inert antibody (P3); IC)PNH I and PNH 111 cells; Id) discrete populations of PNH 1, PNH It, and PNH 111 cells; (e) PNH 1 and PNHII cells; (f) nearly entirely PNH 111 cells. From www.bloodjournal.org by guest on August 10, 2017. For personal use only. DIAGNOSIS OF PNH BY FLOW CYTOMETRY Fig 6. The mean fluorescence channel ofanti-CD59 on granulocytes. [ : I , normal granulocytes; i.’. , inert antibody (P31 controls; 0,PNH I cells; B, PNH It; W, PNH 111 cells. 5337 L 50 Mean Fluorescence Channel The cells of intermediate abnormality (PNH I1 cells) were more difficult to demonstrate than in red cells. PNH I1 cells were not detected by anti-CD16, and the proportion of patients having PNH I1 granulocytes (demonstrated by antiCD55 or anti-CD59) was less than that having PNH I1 erythrocytes. Two techniques were used to prepare the granulocytes: the unseparatedgranulocyte technique, in which the red 8 8 8 8 8 m 8 : m ~ “0 100 20 40 60 80 Percent Abnormal RBC’s 100 Fig 7. A comparison of the proportion of abnormal (PNH II + PNH 111 cells) erythrocytes and the proportion of abnormal granulocytes in the blood of 35 patients with PNH. cells of whole blood were hemolyzed and the granulocytes reacted with the antibody and analyzed, and the separated granulocyte technique, in which the granulocytes were separated from other blood elements by differential centrifugation before testing. Whereas abnormal granulocytes could always be detected in the simpler unseparated assay, the distinctions between different populations and the proportions of cells in each population were less clear than in the tests using separated granulocytes. With both tests, a small number of “granulocytes” in normalblooddidnot stain with the antibody; as with the red cells, this was not consistent in a given patient and was thought to be artifactual. However, the number of cells in normal blood that did not bind the antibody was very muchhigher with the unseparated granulocyte technique; for this reason, that technique, although simpler, should be used with caution in the analysis of blood for the diagnosis of PNH, particularly whenthe proportion of abnormal cells is low. Samples older than 2 days could not be used for analysis because of poor quality of the cells. In general, the proportion of abnormal granulocytes detected was greater than the number of abnormal red cells from the same blood sample. This observation has been previously found by flow cytometry and other techniques.I8The explanation lies in part in the fact that the survival in the circulation of the abnormal granulocytes appears to be normal,’’ whereas that of the abnormal red cells is reduced.2” Thus, the percentage of circulating abnormal granulocytes more nearly represents the proportion of abnormal cells delivered to the circulation. Cells with less thannormal amounts of the GPI-linked proteins (PNH I1 cells) were demonstrated in both erythrocytes and granulocytes in a majority of cases when anti- From www.bloodjournal.org by guest on August 10, 2017. For personal use only. 5338 HALL AND ROSSE CD59 was used for detection. These cells were less easily seen with anti-CD55 and were not observed in granulocytes with anti-CD16. In some cases, the partial deficiency is due to a missense mutation of the pig-A gene2',22;in others, the identified defect appears to be one thatwouldleadto a premature stop codon.22The reason for this discrepancy is under investigation. The deficiency of the GPI-dependent proteins couldbe demonstrated in the platelets of patients with PNH but the fluorescence of normal and PNH I platelets was little different from the negative control and the distinction between PNH I and PNH I11 platelets was difficult and imprecise. GPI-dependent protein deficient lymphocytes are demonstrable in most ifall patients not with PNH'Z.'h,23-26. ,Immortalized lymphocyte cell lines have played an important role in the identification of the genetic defect in PNH.27-2y In most patients, the proportion of abnormal lymphocytes is small, particularly relative to the proportion of abnormal erythrocytes or granulocytes in the same blood. In some studies, the abnormal lymphocytes were missed entirely if they were in fact present.'' Thus, analysis of lymphocytes for GPIlinked proteins to make the diagnosis of PNH is not recommended. On the other hand, in patients who recover from PNH with disappearance of abnormal erythrocytes and granulocytes may have a persistent population of GPI-deficient lymphocytes." This is thought to be due to the longevity of lymphocytes compared with other blood cells. How cytometry andMoAbsto GPI-dependent proteins have been used to analyze the cells of the bone marrow in PNH. In general, the proportion of abnormal cells corresponds roughly to that of the granulocytes. In exceptional cases, the abnormal cells in the bone marrow can bedetected in the marrow for several months before they appear in the peripheral blood.3' Small populations of cells deficient in GPI-dependent proteins can be demonstrated in the blood of 15% to 30% of patients with aplastic anemia, particularly after treatment with antithymocyte These populations are diagnostic of PNH by definition, but the clinical syndrome often remains that of aplastic anemia. These findings indicate a close but not clearly understood relationship between the two ~yndromes.'~,~' These studies clearly demonstrate the utility of MoAbs and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Flow cytometry is shown in these studies to be superior to the CLS assay in that small populations of abnormal cells may be missedoccasionally in the CLS test (1 patient in this series). Further, flow cytometry distinguishes much better the cells of intermediate abnormality (PNH I1 cells); while this may not be of great interest diagnostically, it is of major importance in characterizing the abnormalities in the cells of different patients. We have previously shown that the CLS test was superior to either the Ham test or the sucrose lysis test?' Even when the Hamtest is done well, it does not detect small populations of abnormal cells (which results in a decrease in sensitivity), the degree of lysis does not accurately reflect the proportion of abnormal cells,"' and the degree of abnormality of the ' abnormal cells cannot be assessed. Furthermore, the test is falsely positive in the rare disorder hereditary erythrocytic multinuclearity with a positive acidified-serum lysistest ([HEMPAS] congenital dyserythropoietic anemia, type H).''' The sucrose lysis test is technically easier to performbut has a higher rate of false-positive resultsu (thus decreasing its specificity). Although it more accurately detects the proportion of PNH 111 red cells than the Ham test, it likewise does not delineate the abnormality of these cells and does not quantitatively delineate the number of PNH I1 cells."' Flow cytometry has, in addition, the advantage that the reagents can be standardized; the tests depending upon hemolysis use normal human serum as a reagent and there may be great variability in the potency of normal serum to lyse the abnormal red cells.'s Further, the reproducibility of the results from flow cytometry is greater thanthat of either other test. Red cells can be stored up to 3 weeks without change in the characteristics of the cells whereas granulocytes are difficult to analyze if they are more than 12 to 24 hours old. The estimated costs of this more accurate diagnostic technique may be 1 to 1.5 times as much as the conventional but less accurate serological tests. Flow cytometry in the diagnosis of PNH is specific in that no other clinical syndrome is characterized by cells with the phenotype demonstrated by this technique. Hematopoietic cells congenitally deficient inCD5546.47and CD594Rhave been described, but these extremely rare conditions are readily distinguished from PNH by the facts that 100% of the cells are deficient in these syndromes and only one of the proteins is deficient. Because flow cytometry is able to demonstrate the characteristic cells with greater certainty and precision than previous methods using secondary effects (eg, increased sensitivity to complement lysis), we propose that the analysis byflow cytometry of erythrocytes using antiCD59 or of granulocytes using either anti-CD59 or antiCD55 be the standard diagnostic test for paroxysmal nocturnal hemoglobinuria. REFERENCES 1. Rosse WF: Phosphatidylinositol-linked proteins and paroxysmal nocturnal hemoglobinuria. Blood 75: 1595, 1990 2. Yeh ETH, Rosse WF: Paroxysmal nocturnal hemoglobinuria and the glycosylphosphatidylinositol anchor. J Clin Invest 93:2305, 1994 3. Takeda J, Miyata T, Kawagoe K, Iida Y , Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T: Deficiency of the GP1 anchor caused by a somatic mutation of thePIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 73:703, 1993 4. Holguin MH, Frederick LR, Bernshaw NJ, Wilcox LA, Parker CJ: Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria. J Clin Invest 84:7, 1989 5. Men S, Waldmann H, Lachmann PJ: Distribution of protectin (CD59), a complement membrane attack inhibitor, in normal human tissues. Lab Invest 65532, 1991 6. Davies A, Simmons DL, Hale G , Harrison RA, Tighe H, Lachmann PJ, Waldmann H: CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement mem- From www.bloodjournal.org by guest on August 10, 2017. For personal use only. DIAGNOSIS OF PNH BY FLOW CMOMETRY brane attack complex on homologous cells. J Exp Med 170:637, 1989 7. Sugita Y, Nakano Y, Oda E, Noda K, Tobe T, Miura NH, Tomita M . Determination of carboxyl-terminal residue and disulfide bonds of MACE (CD59), a glycosyl-phosphatidylinositol-anchored membrane protein. J Biochem (Tokyo) 114:473, 1993 8. Nicholson-Weller A, March JP, Rosenfeld SI, Austen KF: Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in the complement regulatory protein, decay accelerating factor. Proc Natl Acad Sci USA 805430, 1983 9. Rosse WF, Dacie JV: Immune lysis of normal human and paroxysmal nocturnal hemoglobinuria red blood cells. I. 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Rosse WF, Hoffman S, Campbell M, Borowitz M, Moore JO, Parker CJ: The erythrocytes in paroxysmal nocturnal haemoglobinuria of intermediate sensitivity to complement lysis. Br J Haemato1 79:99, 1991 14. Shichishima T, Terasawa T, Hashimoto C, Ohto H, Uchida T, Maruyama Y: Heterogenous expression of decay accelerating factor and CD59/membrane attack complex inhibition factor on paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes. Br J Haematol 78:545, 1991 15. Shichishima T, Terasawa T, Saitoh Y, Hashimoto C, Ohto H, Maruyama A: Diagnosis of paroxysmal nocturnal haemoglobinuria by phenotypic analysis of erythrocytes using two-color flow cytometry with monoclonal antibodies to DAF and CD59IMACIF. Br J Haematol 85:378, 1993 16. Kwong YL, Lee CP, Chan TK, Chan LC: Flow cytometric measurement of glycosylphosphatidyl-inositol-linked surface proteins on blood cells of patients with paroxysmal nocturnal hemoglobinuria. Am J Clin Pathol 102:30, 1994 17. 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For personal use only. 1996 87: 5332-5340 The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria SE Hall and WF Rosse Updated information and services can be found at: http://www.bloodjournal.org/content/87/12/5332.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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